Difference between revisions of "Team:HK SSC/Notebook"

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{{HK_SSC}}
 
{{HK_SSC}}
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  <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
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</w:LatentStyles>
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/* Style Definitions */
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p.MsoNormal, li.MsoNormal, div.MsoNormal
 +
{mso-style-unhide:no;
 +
mso-style-qformat:yes;
 +
mso-style-parent:"";
 +
margin:0cm;
 +
margin-bottom:.0001pt;
 +
mso-pagination:widow-orphan;
 +
font-size:12.0pt;
 +
font-family:"Calibri",sans-serif;
 +
mso-ascii-font-family:Calibri;
 +
mso-ascii-theme-font:minor-latin;
 +
mso-fareast-font-family:PMingLiU;
 +
mso-fareast-theme-font:minor-fareast;
 +
mso-hansi-font-family:Calibri;
 +
mso-hansi-theme-font:minor-latin;
 +
mso-bidi-font-family:Arial;
 +
mso-bidi-theme-font:minor-bidi;}
 +
p.MsoAcetate, li.MsoAcetate, div.MsoAcetate
 +
{mso-style-noshow:yes;
 +
mso-style-priority:99;
 +
mso-style-link:"Balloon Text Char";
 +
margin:0cm;
 +
margin-bottom:.0001pt;
 +
mso-pagination:widow-orphan;
 +
font-size:9.0pt;
 +
font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:PMingLiU;
 +
mso-fareast-theme-font:minor-fareast;}
 +
p.MsoRMPane, li.MsoRMPane, div.MsoRMPane
 +
{mso-style-noshow:yes;
 +
mso-style-priority:99;
 +
mso-style-unhide:no;
 +
mso-style-parent:"";
 +
margin:0cm;
 +
margin-bottom:.0001pt;
 +
mso-pagination:widow-orphan;
 +
font-size:12.0pt;
 +
font-family:"Calibri",sans-serif;
 +
mso-ascii-font-family:Calibri;
 +
mso-ascii-theme-font:minor-latin;
 +
mso-fareast-font-family:PMingLiU;
 +
mso-fareast-theme-font:minor-fareast;
 +
mso-hansi-font-family:Calibri;
 +
mso-hansi-theme-font:minor-latin;
 +
mso-bidi-font-family:Arial;
 +
mso-bidi-theme-font:minor-bidi;}
 +
p.MsoListParagraph, li.MsoListParagraph, div.MsoListParagraph
 +
{mso-style-priority:34;
 +
mso-style-unhide:no;
 +
mso-style-qformat:yes;
 +
margin-top:0cm;
 +
margin-right:0cm;
 +
margin-bottom:0cm;
 +
margin-left:36.0pt;
 +
margin-bottom:.0001pt;
 +
mso-add-space:auto;
 +
mso-pagination:widow-orphan;
 +
font-size:12.0pt;
 +
font-family:"Calibri",sans-serif;
 +
mso-ascii-font-family:Calibri;
 +
mso-ascii-theme-font:minor-latin;
 +
mso-fareast-font-family:PMingLiU;
 +
mso-fareast-theme-font:minor-fareast;
 +
mso-hansi-font-family:Calibri;
 +
mso-hansi-theme-font:minor-latin;
 +
mso-bidi-font-family:Arial;
 +
mso-bidi-theme-font:minor-bidi;}
 +
p.MsoListParagraphCxSpFirst, li.MsoListParagraphCxSpFirst, div.MsoListParagraphCxSpFirst
 +
{mso-style-priority:34;
 +
mso-style-unhide:no;
 +
mso-style-qformat:yes;
 +
mso-style-type:export-only;
 +
margin-top:0cm;
 +
margin-right:0cm;
 +
margin-bottom:0cm;
 +
margin-left:36.0pt;
 +
margin-bottom:.0001pt;
 +
mso-add-space:auto;
 +
mso-pagination:widow-orphan;
 +
font-size:12.0pt;
 +
font-family:"Calibri",sans-serif;
 +
mso-ascii-font-family:Calibri;
 +
mso-ascii-theme-font:minor-latin;
 +
mso-fareast-font-family:PMingLiU;
 +
mso-fareast-theme-font:minor-fareast;
 +
mso-hansi-font-family:Calibri;
 +
mso-hansi-theme-font:minor-latin;
 +
mso-bidi-font-family:Arial;
 +
mso-bidi-theme-font:minor-bidi;}
 +
p.MsoListParagraphCxSpMiddle, li.MsoListParagraphCxSpMiddle, div.MsoListParagraphCxSpMiddle
 +
{mso-style-priority:34;
 +
mso-style-unhide:no;
 +
mso-style-qformat:yes;
 +
mso-style-type:export-only;
 +
margin-top:0cm;
 +
margin-right:0cm;
 +
margin-bottom:0cm;
 +
margin-left:36.0pt;
 +
margin-bottom:.0001pt;
 +
mso-add-space:auto;
 +
mso-pagination:widow-orphan;
 +
font-size:12.0pt;
 +
font-family:"Calibri",sans-serif;
 +
mso-ascii-font-family:Calibri;
 +
mso-ascii-theme-font:minor-latin;
 +
mso-fareast-font-family:PMingLiU;
 +
mso-fareast-theme-font:minor-fareast;
 +
mso-hansi-font-family:Calibri;
 +
mso-hansi-theme-font:minor-latin;
 +
mso-bidi-font-family:Arial;
 +
mso-bidi-theme-font:minor-bidi;}
 +
p.MsoListParagraphCxSpLast, li.MsoListParagraphCxSpLast, div.MsoListParagraphCxSpLast
 +
{mso-style-priority:34;
 +
mso-style-unhide:no;
 +
mso-style-qformat:yes;
 +
mso-style-type:export-only;
 +
margin-top:0cm;
 +
margin-right:0cm;
 +
margin-bottom:0cm;
 +
margin-left:36.0pt;
 +
margin-bottom:.0001pt;
 +
mso-add-space:auto;
 +
mso-pagination:widow-orphan;
 +
font-size:12.0pt;
 +
font-family:"Calibri",sans-serif;
 +
mso-ascii-font-family:Calibri;
 +
mso-ascii-theme-font:minor-latin;
 +
mso-fareast-font-family:PMingLiU;
 +
mso-fareast-theme-font:minor-fareast;
 +
mso-hansi-font-family:Calibri;
 +
mso-hansi-theme-font:minor-latin;
 +
mso-bidi-font-family:Arial;
 +
mso-bidi-theme-font:minor-bidi;}
 +
span.BalloonTextChar
 +
{mso-style-name:"Balloon Text Char";
 +
mso-style-noshow:yes;
 +
mso-style-priority:99;
 +
mso-style-unhide:no;
 +
mso-style-locked:yes;
 +
mso-style-link:"Balloon Text";
 +
mso-ansi-font-size:9.0pt;
 +
mso-bidi-font-size:9.0pt;
 +
font-family:"Times New Roman",serif;
 +
mso-ascii-font-family:"Times New Roman";
 +
mso-hansi-font-family:"Times New Roman";
 +
mso-bidi-font-family:"Times New Roman";}
 +
span.SpellE
 +
{mso-style-name:"";
 +
mso-spl-e:yes;}
 +
span.GramE
 +
{mso-style-name:"";
 +
mso-gram-e:yes;}
 +
.MsoChpDefault
 +
{mso-style-type:export-only;
 +
mso-default-props:yes;
 +
font-family:"Calibri",sans-serif;
 +
mso-ascii-font-family:Calibri;
 +
mso-ascii-theme-font:minor-latin;
 +
mso-fareast-font-family:PMingLiU;
 +
mso-fareast-theme-font:minor-fareast;
 +
mso-hansi-font-family:Calibri;
 +
mso-hansi-theme-font:minor-latin;
 +
mso-bidi-font-family:Arial;
 +
mso-bidi-theme-font:minor-bidi;}
 +
@page WordSection1
 +
{size:595.0pt 842.0pt;
 +
margin:72.0pt 72.0pt 72.0pt 72.0pt;
 +
mso-header-margin:35.4pt;
 +
mso-footer-margin:35.4pt;
 +
mso-paper-source:0;}
 +
div.WordSection1
 +
{page:WordSection1;}
 +
/* List Definitions */
 +
@list l0
 +
{mso-list-id:128939763;
 +
mso-list-type:hybrid;
 +
mso-list-template-ids:-1 764586504 -1933031626 1761646812 -947849546 1804654132 278010046 2089826584 -1585517318 -1021528830;}
 +
@list l0:level1
 +
{mso-level-number-format:bullet;
 +
mso-level-text:;
 +
mso-level-tab-stop:none;
 +
mso-level-number-position:left;
 +
text-indent:-18.0pt;
 +
font-family:Symbol;}
 +
@list l0:level2
 +
{mso-level-number-format:bullet;
 +
mso-level-text:o;
 +
mso-level-tab-stop:none;
 +
mso-level-number-position:left;
 +
text-indent:-18.0pt;
 +
font-family:"Courier New";
 +
mso-bidi-font-family:"Times New Roman";}
 +
@list l0:level3
 +
{mso-level-number-format:bullet;
 +
mso-level-text:;
 +
mso-level-tab-stop:none;
 +
mso-level-number-position:left;
 +
text-indent:-18.0pt;
 +
font-family:Wingdings;}
 +
@list l0:level4
 +
{mso-level-number-format:bullet;
 +
mso-level-text:;
 +
mso-level-tab-stop:none;
 +
mso-level-number-position:left;
 +
text-indent:-18.0pt;
 +
font-family:Symbol;}
 +
@list l0:level5
 +
{mso-level-number-format:bullet;
 +
mso-level-text:o;
 +
mso-level-tab-stop:none;
 +
mso-level-number-position:left;
 +
text-indent:-18.0pt;
 +
font-family:"Courier New";
 +
mso-bidi-font-family:"Times New Roman";}
 +
@list l0:level6
 +
{mso-level-number-format:bullet;
 +
mso-level-text:;
 +
mso-level-tab-stop:none;
 +
mso-level-number-position:left;
 +
text-indent:-18.0pt;
 +
font-family:Wingdings;}
 +
@list l0:level7
 +
{mso-level-number-format:bullet;
 +
mso-level-text:;
 +
mso-level-tab-stop:none;
 +
mso-level-number-position:left;
 +
text-indent:-18.0pt;
 +
font-family:Symbol;}
 +
@list l0:level8
 +
{mso-level-number-format:bullet;
 +
mso-level-text:o;
 +
mso-level-tab-stop:none;
 +
mso-level-number-position:left;
 +
text-indent:-18.0pt;
 +
font-family:"Courier New";
 +
mso-bidi-font-family:"Times New Roman";}
 +
@list l0:level9
 +
{mso-level-number-format:bullet;
 +
mso-level-text:;
 +
mso-level-tab-stop:none;
 +
mso-level-number-position:left;
 +
text-indent:-18.0pt;
 +
font-family:Wingdings;}
 +
@list l1
 +
{mso-list-id:135998541;
 +
mso-list-type:hybrid;
 +
mso-list-template-ids:-516132334 134807567 134807577 134807579 134807567 134807577 134807579 134807567 134807577 134807579;}
 +
@list l1:level1
 +
{mso-level-tab-stop:none;
 +
mso-level-number-position:left;
 +
text-indent:-18.0pt;}
 +
@list l1:level2
 +
{mso-level-number-format:alpha-lower;
 +
mso-level-tab-stop:none;
 +
mso-level-number-position:left;
 +
text-indent:-18.0pt;}
 +
@list l1:level3
 +
{mso-level-number-format:roman-lower;
 +
mso-level-tab-stop:none;
 +
mso-level-number-position:right;
 +
text-indent:-9.0pt;}
 +
@list l1:level4
 +
{mso-level-tab-stop:none;
 +
mso-level-number-position:left;
 +
text-indent:-18.0pt;}
 +
@list l1:level5
 +
{mso-level-number-format:alpha-lower;
 +
mso-level-tab-stop:none;
 +
mso-level-number-position:left;
 +
text-indent:-18.0pt;}
 +
@list l1:level6
 +
{mso-level-number-format:roman-lower;
 +
mso-level-tab-stop:none;
 +
mso-level-number-position:right;
 +
text-indent:-9.0pt;}
 +
@list l1:level7
 +
{mso-level-tab-stop:none;
 +
mso-level-number-position:left;
 +
text-indent:-18.0pt;}
 +
@list l1:level8
 +
{mso-level-number-format:alpha-lower;
 +
mso-level-tab-stop:none;
 +
mso-level-number-position:left;
 +
text-indent:-18.0pt;}
 +
@list l1:level9
 +
{mso-level-number-format:roman-lower;
 +
mso-level-tab-stop:none;
 +
mso-level-number-position:right;
 +
text-indent:-9.0pt;}
 +
@list l2
 +
{mso-list-id:163588628;
 +
mso-list-type:hybrid;
 +
mso-list-template-ids:2107543474 134807569 134807577 134807579 134807567 134807577 134807579 134807567 134807577 134807579;}
 +
@list l2:level1
 +
{mso-level-text:"%1\)";
 +
mso-level-tab-stop:none;
 +
mso-level-number-position:left;
 +
text-indent:-18.0pt;}
 +
@list l2:level2
 +
{mso-level-number-format:alpha-lower;
 +
mso-level-tab-stop:none;
 +
mso-level-number-position:left;
 +
text-indent:-18.0pt;}
 +
@list l2:level3
 +
{mso-level-number-format:roman-lower;
 +
mso-level-tab-stop:none;
 +
mso-level-number-position:right;
 +
text-indent:-9.0pt;}
 +
@list l2:level4
 +
{mso-level-tab-stop:none;
 +
mso-level-number-position:left;
 +
text-indent:-18.0pt;}
 +
@list l2:level5
 +
{mso-level-number-format:alpha-lower;
 +
mso-level-tab-stop:none;
 +
mso-level-number-position:left;
 +
text-indent:-18.0pt;}
 +
@list l2:level6
 +
{mso-level-number-format:roman-lower;
 +
mso-level-tab-stop:none;
 +
mso-level-number-position:right;
 +
text-indent:-9.0pt;}
 +
@list l2:level7
 +
{mso-level-tab-stop:none;
 +
mso-level-number-position:left;
 +
text-indent:-18.0pt;}
 +
@list l2:level8
 +
{mso-level-number-format:alpha-lower;
 +
mso-level-tab-stop:none;
 +
mso-level-number-position:left;
 +
text-indent:-18.0pt;}
 +
@list l2:level9
 +
{mso-level-number-format:roman-lower;
 +
mso-level-tab-stop:none;
 +
mso-level-number-position:right;
 +
text-indent:-9.0pt;}
 +
@list l3
 +
{mso-list-id:371804689;
 +
mso-list-type:hybrid;
 +
mso-list-template-ids:-1 1130762128 1784311152 -733452568 1603929906 1277692258 805210230 -1163461986 861171978 188898512;}
 +
@list l3:level1
 +
{mso-level-number-format:bullet;
 +
mso-level-text:;
 +
mso-level-tab-stop:none;
 +
mso-level-number-position:left;
 +
text-indent:-18.0pt;
 +
font-family:Symbol;}
 +
@list l3:level2
 +
{mso-level-number-format:bullet;
 +
mso-level-text:o;
 +
mso-level-tab-stop:none;
 +
mso-level-number-position:left;
 +
text-indent:-18.0pt;
 +
font-family:"Courier New";
 +
mso-bidi-font-family:"Times New Roman";}
 +
@list l3:level3
 +
{mso-level-number-format:bullet;
 +
mso-level-text:;
 +
mso-level-tab-stop:none;
 +
mso-level-number-position:left;
 +
text-indent:-18.0pt;
 +
font-family:Wingdings;}
 +
@list l3:level4
 +
{mso-level-number-format:bullet;
 +
mso-level-text:;
 +
mso-level-tab-stop:none;
 +
mso-level-number-position:left;
 +
text-indent:-18.0pt;
 +
font-family:Symbol;}
 +
@list l3:level5
 +
{mso-level-number-format:bullet;
 +
mso-level-text:o;
 +
mso-level-tab-stop:none;
 +
mso-level-number-position:left;
 +
text-indent:-18.0pt;
 +
font-family:"Courier New";
 +
mso-bidi-font-family:"Times New Roman";}
 +
@list l3:level6
 +
{mso-level-number-format:bullet;
 +
mso-level-text:;
 +
mso-level-tab-stop:none;
 +
mso-level-number-position:left;
 +
text-indent:-18.0pt;
 +
font-family:Wingdings;}
 +
@list l3:level7
 +
{mso-level-number-format:bullet;
 +
mso-level-text:;
 +
mso-level-tab-stop:none;
 +
mso-level-number-position:left;
 +
text-indent:-18.0pt;
 +
font-family:Symbol;}
 +
@list l3:level8
 +
{mso-level-number-format:bullet;
 +
mso-level-text:o;
 +
mso-level-tab-stop:none;
 +
mso-level-number-position:left;
 +
text-indent:-18.0pt;
 +
font-family:"Courier New";
 +
mso-bidi-font-family:"Times New Roman";}
 +
@list l3:level9
 +
{mso-level-number-format:bullet;
 +
mso-level-text:;
 +
mso-level-tab-stop:none;
 +
mso-level-number-position:left;
 +
text-indent:-18.0pt;
 +
font-family:Wingdings;}
 +
@list l4
 +
{mso-list-id:382564340;
 +
mso-list-type:hybrid;
 +
mso-list-template-ids:-1 892638038 -492403742 -250338136 -881064854 -917996576 -872377190 1871725450 -1683873958 865642644;}
 +
@list l4:level1
 +
{mso-level-number-format:bullet;
 +
mso-level-text:;
 +
mso-level-tab-stop:none;
 +
mso-level-number-position:left;
 +
text-indent:-18.0pt;
 +
font-family:Symbol;}
 +
@list l4:level2
 +
{mso-level-number-format:bullet;
 +
mso-level-text:o;
 +
mso-level-tab-stop:none;
 +
mso-level-number-position:left;
 +
text-indent:-18.0pt;
 +
font-family:"Courier New";
 +
mso-bidi-font-family:"Times New Roman";}
 +
@list l4:level3
 +
{mso-level-number-format:bullet;
 +
mso-level-text:;
 +
mso-level-tab-stop:none;
 +
mso-level-number-position:left;
 +
text-indent:-18.0pt;
 +
font-family:Wingdings;}
 +
@list l4:level4
 +
{mso-level-number-format:bullet;
 +
mso-level-text:;
 +
mso-level-tab-stop:none;
 +
mso-level-number-position:left;
 +
text-indent:-18.0pt;
 +
font-family:Symbol;}
 +
@list l4:level5
 +
{mso-level-number-format:bullet;
 +
mso-level-text:o;
 +
mso-level-tab-stop:none;
 +
mso-level-number-position:left;
 +
text-indent:-18.0pt;
 +
font-family:"Courier New";
 +
mso-bidi-font-family:"Times New Roman";}
 +
@list l4:level6
 +
{mso-level-number-format:bullet;
 +
mso-level-text:;
 +
mso-level-tab-stop:none;
 +
mso-level-number-position:left;
 +
text-indent:-18.0pt;
 +
font-family:Wingdings;}
 +
@list l4:level7
 +
{mso-level-number-format:bullet;
 +
mso-level-text:;
 +
mso-level-tab-stop:none;
 +
mso-level-number-position:left;
 +
text-indent:-18.0pt;
 +
font-family:Symbol;}
 +
@list l4:level8
 +
{mso-level-number-format:bullet;
 +
mso-level-text:o;
 +
mso-level-tab-stop:none;
 +
mso-level-number-position:left;
 +
text-indent:-18.0pt;
 +
font-family:"Courier New";
 +
mso-bidi-font-family:"Times New Roman";}
 +
@list l4:level9
 +
{mso-level-number-format:bullet;
 +
mso-level-text:;
 +
mso-level-tab-stop:none;
 +
mso-level-number-position:left;
 +
text-indent:-18.0pt;
 +
font-family:Wingdings;}
 +
@list l5
 +
{mso-list-id:388188240;
 +
mso-list-type:hybrid;
 +
mso-list-template-ids:-531336032 -1363879744 134807555 134807557 134807553 134807555 134807557 134807553 134807555 134807557;}
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@list l5:level1
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{mso-level-start-at:9;
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mso-level-number-format:bullet;
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mso-level-text:;
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mso-level-tab-stop:none;
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mso-level-number-position:left;
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margin-left:53.45pt;
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font-family:Wingdings;
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mso-fareast-font-family:PMingLiU;
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mso-fareast-theme-font:minor-fareast;
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mso-bidi-font-family:Arial;
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mso-bidi-theme-font:minor-bidi;}
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@list l5:level2
 +
{mso-level-number-format:bullet;
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mso-level-text:o;
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mso-level-tab-stop:none;
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mso-level-number-position:left;
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margin-left:89.45pt;
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text-indent:-18.0pt;
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font-family:"Courier New";}
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@list l5:level3
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{mso-level-number-format:bullet;
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mso-level-text:;
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mso-level-tab-stop:none;
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mso-level-number-position:left;
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margin-left:125.45pt;
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text-indent:-18.0pt;
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font-family:Wingdings;}
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@list l5:level4
 +
{mso-level-number-format:bullet;
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mso-level-text:;
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mso-level-tab-stop:none;
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mso-level-number-position:left;
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margin-left:161.45pt;
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text-indent:-18.0pt;
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font-family:Symbol;}
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@list l5:level5
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{mso-level-number-format:bullet;
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mso-level-text:o;
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mso-level-tab-stop:none;
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mso-level-number-position:left;
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margin-left:197.45pt;
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text-indent:-18.0pt;
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font-family:"Courier New";}
 +
@list l5:level6
 +
{mso-level-number-format:bullet;
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mso-level-text:;
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mso-level-tab-stop:none;
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mso-level-number-position:left;
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margin-left:233.45pt;
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text-indent:-18.0pt;
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font-family:Wingdings;}
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@list l5:level7
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{mso-level-number-format:bullet;
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mso-level-text:;
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mso-level-tab-stop:none;
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mso-level-number-position:left;
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margin-left:269.45pt;
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text-indent:-18.0pt;
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font-family:Symbol;}
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@list l5:level8
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{mso-level-number-format:bullet;
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mso-level-text:o;
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mso-level-tab-stop:none;
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mso-level-number-position:left;
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margin-left:305.45pt;
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text-indent:-18.0pt;
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font-family:"Courier New";}
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@list l5:level9
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{mso-level-number-format:bullet;
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mso-level-text:;
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mso-level-tab-stop:none;
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mso-level-number-position:left;
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margin-left:341.45pt;
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font-family:Wingdings;}
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@list l6
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{mso-list-id:402260799;
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mso-list-template-ids:-1237158604 134807569 134807577 134807579 134807567 134807577 134807579 134807567 134807577 134807579;}
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@list l6:level1
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{mso-level-text:"%1\)";
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mso-level-tab-stop:none;
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mso-level-number-position:left;
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text-indent:-18.0pt;}
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@list l6:level2
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{mso-level-number-format:alpha-lower;
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mso-level-tab-stop:none;
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mso-level-number-position:left;
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text-indent:-18.0pt;}
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@list l6:level3
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{mso-level-number-format:roman-lower;
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mso-level-tab-stop:none;
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mso-level-number-position:right;
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text-indent:-9.0pt;}
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@list l6:level4
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{mso-level-tab-stop:none;
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mso-level-number-position:left;
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text-indent:-18.0pt;}
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@list l6:level5
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{mso-level-number-format:alpha-lower;
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mso-level-tab-stop:none;
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mso-level-number-position:left;
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text-indent:-18.0pt;}
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@list l6:level6
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{mso-level-number-format:roman-lower;
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mso-level-tab-stop:none;
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mso-level-number-position:right;
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text-indent:-9.0pt;}
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@list l6:level7
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{mso-level-tab-stop:none;
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mso-level-number-position:left;
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text-indent:-18.0pt;}
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@list l6:level8
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{mso-level-number-format:alpha-lower;
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mso-level-tab-stop:none;
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mso-level-number-position:left;
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text-indent:-18.0pt;}
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@list l6:level9
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{mso-level-number-format:roman-lower;
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mso-level-tab-stop:none;
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mso-level-number-position:right;
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text-indent:-9.0pt;}
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@list l7
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{mso-list-id:428162354;
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mso-list-template-ids:-1 -1252733568 930100114 -557006734 1384544668 -954848828 -1571635198 -1542271392 -424786644 15750658;}
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@list l7:level1
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{mso-level-number-format:bullet;
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mso-level-text:;
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mso-level-tab-stop:none;
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mso-level-number-position:left;
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text-indent:-18.0pt;
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font-family:Symbol;}
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@list l7:level2
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{mso-level-number-format:bullet;
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mso-level-text:o;
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mso-level-tab-stop:none;
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mso-level-number-position:left;
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text-indent:-18.0pt;
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font-family:"Courier New";
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mso-bidi-font-family:"Times New Roman";}
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@list l7:level3
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{mso-level-number-format:bullet;
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mso-level-text:;
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mso-level-tab-stop:none;
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mso-level-number-position:left;
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text-indent:-18.0pt;
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font-family:Wingdings;}
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@list l7:level4
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{mso-level-number-format:bullet;
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mso-level-tab-stop:none;
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mso-level-number-position:left;
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text-indent:-18.0pt;
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font-family:Symbol;}
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@list l7:level5
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{mso-level-number-format:bullet;
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mso-level-text:o;
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mso-level-tab-stop:none;
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mso-level-number-position:left;
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text-indent:-18.0pt;
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font-family:"Courier New";
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mso-bidi-font-family:"Times New Roman";}
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@list l7:level6
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{mso-level-number-format:bullet;
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mso-level-text:;
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mso-level-tab-stop:none;
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mso-level-number-position:left;
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text-indent:-18.0pt;
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font-family:Wingdings;}
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@list l7:level7
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{mso-level-number-format:bullet;
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mso-level-text:;
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mso-level-tab-stop:none;
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mso-level-number-position:left;
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text-indent:-18.0pt;
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font-family:Symbol;}
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@list l7:level8
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{mso-level-number-format:bullet;
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mso-level-text:o;
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mso-level-tab-stop:none;
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mso-level-number-position:left;
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text-indent:-18.0pt;
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font-family:"Courier New";
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mso-bidi-font-family:"Times New Roman";}
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@list l7:level9
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{mso-level-number-format:bullet;
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mso-level-text:;
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mso-level-tab-stop:none;
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mso-level-number-position:left;
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text-indent:-18.0pt;
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font-family:Wingdings;}
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{mso-list-id:483546897;
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mso-list-template-ids:-1 -1060080912 -637471564 605867450 331887558 -1175790214 1068922962 1718492644 525772238 1941732984;}
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@list l8:level1
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{mso-level-number-format:bullet;
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mso-level-text:;
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mso-level-tab-stop:none;
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mso-level-number-position:left;
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text-indent:-18.0pt;
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font-family:Symbol;}
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@list l8:level2
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{mso-level-number-format:bullet;
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mso-level-text:o;
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mso-level-tab-stop:none;
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mso-level-number-position:left;
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text-indent:-18.0pt;
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font-family:"Courier New";
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mso-bidi-font-family:"Times New Roman";}
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@list l8:level3
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{mso-level-number-format:bullet;
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mso-level-text:;
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mso-level-tab-stop:none;
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mso-level-number-position:left;
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text-indent:-18.0pt;
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font-family:Wingdings;}
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@list l8:level4
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{mso-level-number-format:bullet;
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mso-level-text:;
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mso-level-tab-stop:none;
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mso-level-number-position:left;
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text-indent:-18.0pt;
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font-family:Symbol;}
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@list l8:level5
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{mso-level-number-format:bullet;
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mso-level-text:o;
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mso-level-tab-stop:none;
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mso-level-number-position:left;
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text-indent:-18.0pt;
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font-family:"Courier New";
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mso-bidi-font-family:"Times New Roman";}
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@list l8:level6
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{mso-level-number-format:bullet;
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mso-level-text:;
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mso-level-tab-stop:none;
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mso-level-number-position:left;
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text-indent:-18.0pt;
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font-family:Wingdings;}
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@list l8:level7
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{mso-level-number-format:bullet;
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mso-level-text:;
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mso-level-tab-stop:none;
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mso-level-number-position:left;
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text-indent:-18.0pt;
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font-family:Symbol;}
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@list l8:level8
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{mso-level-number-format:bullet;
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mso-level-text:o;
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mso-level-tab-stop:none;
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mso-level-number-position:left;
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text-indent:-18.0pt;
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font-family:"Courier New";
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mso-bidi-font-family:"Times New Roman";}
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@list l8:level9
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{mso-level-number-format:bullet;
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mso-level-text:;
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mso-level-tab-stop:none;
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mso-level-number-position:left;
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font-family:Wingdings;}
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@list l9:level1
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{mso-level-number-format:bullet;
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mso-level-text:;
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mso-level-tab-stop:none;
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mso-level-number-position:left;
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text-indent:-18.0pt;
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font-family:Symbol;}
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@list l9:level2
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{mso-level-number-format:bullet;
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mso-level-text:o;
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mso-level-tab-stop:none;
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mso-level-number-position:left;
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text-indent:-18.0pt;
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font-family:"Courier New";
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mso-bidi-font-family:"Times New Roman";}
 +
@list l9:level3
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{mso-level-number-format:bullet;
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mso-level-text:;
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mso-level-tab-stop:none;
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mso-level-number-position:left;
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text-indent:-18.0pt;
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font-family:Wingdings;}
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@list l9:level4
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{mso-level-number-format:bullet;
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mso-level-text:;
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mso-level-tab-stop:none;
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mso-level-number-position:left;
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text-indent:-18.0pt;
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font-family:Symbol;}
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@list l9:level5
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{mso-level-number-format:bullet;
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mso-level-text:o;
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mso-level-tab-stop:none;
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mso-level-number-position:left;
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text-indent:-18.0pt;
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font-family:"Courier New";
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mso-bidi-font-family:"Times New Roman";}
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@list l9:level6
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{mso-level-number-format:bullet;
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mso-level-text:;
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mso-level-tab-stop:none;
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mso-level-number-position:left;
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text-indent:-18.0pt;
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font-family:Wingdings;}
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@list l9:level7
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{mso-level-number-format:bullet;
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mso-level-text:;
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mso-level-tab-stop:none;
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mso-level-number-position:left;
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text-indent:-18.0pt;
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font-family:Symbol;}
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@list l9:level8
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{mso-level-number-format:bullet;
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mso-level-text:o;
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mso-level-tab-stop:none;
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mso-level-number-position:left;
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text-indent:-18.0pt;
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font-family:"Courier New";
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mso-bidi-font-family:"Times New Roman";}
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@list l9:level9
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{mso-level-number-format:bullet;
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mso-level-text:;
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mso-level-tab-stop:none;
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mso-level-number-position:left;
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text-indent:-18.0pt;
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font-family:Wingdings;}
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{mso-list-id:556818120;
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mso-list-type:hybrid;
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mso-list-template-ids:-223817416 134807569 134807577 134807579 134807567 134807577 134807579 134807567 134807577 134807579;}
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@list l10:level1
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{mso-level-text:"%1\)";
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mso-level-tab-stop:none;
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mso-level-number-position:left;
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text-indent:-18.0pt;}
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@list l10:level2
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{mso-level-number-format:alpha-lower;
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mso-level-tab-stop:none;
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mso-level-number-position:left;
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text-indent:-18.0pt;}
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@list l10:level3
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{mso-level-number-format:roman-lower;
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mso-level-tab-stop:none;
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mso-level-number-position:right;
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text-indent:-9.0pt;}
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@list l10:level4
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{mso-level-tab-stop:none;
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mso-level-number-position:left;
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text-indent:-18.0pt;}
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@list l10:level5
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{mso-level-number-format:alpha-lower;
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mso-level-tab-stop:none;
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mso-level-number-position:left;
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text-indent:-18.0pt;}
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@list l10:level6
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{mso-level-number-format:roman-lower;
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mso-level-tab-stop:none;
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mso-level-number-position:right;
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text-indent:-9.0pt;}
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@list l10:level7
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{mso-level-tab-stop:none;
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mso-level-number-position:left;
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text-indent:-18.0pt;}
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@list l10:level8
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{mso-level-number-format:alpha-lower;
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mso-level-tab-stop:none;
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mso-level-number-position:left;
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text-indent:-18.0pt;}
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@list l10:level9
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{mso-level-number-format:roman-lower;
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mso-level-tab-stop:none;
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mso-level-number-position:right;
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text-indent:-9.0pt;}
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{mso-list-id:605159927;
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mso-list-template-ids:-1602461364 785703016 134807555 134807557 134807553 134807555 134807557 134807553 134807555 134807557;}
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@list l11:level1
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{mso-level-start-at:2;
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mso-level-number-format:bullet;
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mso-level-text:-;
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mso-level-tab-stop:none;
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mso-level-number-position:left;
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text-indent:-18.0pt;
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font-family:"Calibri",sans-serif;
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mso-fareast-font-family:PMingLiU;
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mso-fareast-theme-font:minor-fareast;
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mso-bidi-font-family:Arial;
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mso-bidi-theme-font:minor-bidi;}
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@list l11:level2
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{mso-level-number-format:bullet;
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mso-level-text:o;
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mso-level-tab-stop:none;
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mso-level-number-position:left;
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text-indent:-18.0pt;
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font-family:"Courier New";}
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@list l11:level3
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{mso-level-number-format:bullet;
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mso-level-text:;
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mso-level-tab-stop:none;
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mso-level-number-position:left;
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font-family:Wingdings;}
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@list l11:level4
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{mso-level-number-format:bullet;
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mso-level-number-position:left;
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text-indent:-18.0pt;
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font-family:Symbol;}
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@list l11:level5
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{mso-level-number-format:bullet;
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mso-level-text:o;
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mso-level-tab-stop:none;
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mso-level-number-position:left;
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text-indent:-18.0pt;
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font-family:"Courier New";}
 +
@list l11:level6
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{mso-level-number-format:bullet;
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mso-level-text:;
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mso-level-tab-stop:none;
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mso-level-number-position:left;
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font-family:Wingdings;}
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@list l11:level7
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{mso-level-number-format:bullet;
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mso-level-text:;
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mso-level-tab-stop:none;
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font-family:Symbol;}
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@list l11:level8
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{mso-level-number-format:bullet;
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mso-level-tab-stop:none;
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mso-level-number-position:left;
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text-indent:-18.0pt;
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font-family:"Courier New";}
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@list l11:level9
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{mso-level-number-format:bullet;
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mso-level-text:;
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mso-level-number-position:left;
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font-family:Wingdings;}
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text-indent:-18.0pt;
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font-family:Symbol;}
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@list l12:level2
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{mso-level-number-format:bullet;
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mso-level-tab-stop:none;
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mso-level-number-position:left;
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text-indent:-18.0pt;
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font-family:"Courier New";
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mso-bidi-font-family:"Times New Roman";}
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@list l12:level3
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mso-level-number-position:left;
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font-family:Wingdings;}
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@list l12:level4
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{mso-level-number-format:bullet;
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font-family:Symbol;}
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@list l12:level5
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mso-list-template-ids:244241152 134807569 134807577 134807579 134807567 134807577 134807579 134807567 134807577 134807579;}
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@list l21:level1
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{mso-level-text:"%1\)";
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mso-level-tab-stop:none;
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mso-level-number-position:left;
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text-indent:-18.0pt;}
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@list l21:level2
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{mso-level-number-format:alpha-lower;
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mso-level-tab-stop:none;
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mso-level-number-position:left;
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text-indent:-18.0pt;}
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@list l21:level3
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{mso-level-number-format:roman-lower;
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mso-level-tab-stop:none;
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mso-level-number-position:right;
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@list l21:level4
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{mso-level-tab-stop:none;
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mso-level-number-position:left;
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text-indent:-18.0pt;}
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@list l21:level5
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{mso-level-number-format:alpha-lower;
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mso-level-tab-stop:none;
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mso-level-number-position:left;
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text-indent:-18.0pt;}
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@list l21:level6
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{mso-level-number-format:roman-lower;
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mso-level-tab-stop:none;
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mso-level-number-position:right;
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text-indent:-9.0pt;}
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@list l21:level7
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{mso-level-tab-stop:none;
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text-indent:-18.0pt;}
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@list l21:level8
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{mso-level-number-format:alpha-lower;
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mso-level-tab-stop:none;
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mso-level-number-position:left;
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text-indent:-18.0pt;}
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@list l21:level9
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{mso-level-number-format:roman-lower;
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mso-level-tab-stop:none;
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mso-level-number-position:right;
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text-indent:-9.0pt;}
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{mso-list-id:1471169153;
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mso-list-template-ids:-100632800 -873438134 134807555 134807557 134807553 134807555 134807557 134807553 134807555 134807557;}
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@list l22:level1
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{mso-level-start-at:9;
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mso-level-number-format:bullet;
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mso-level-text:-;
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mso-level-tab-stop:none;
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font-family:"Calibri",sans-serif;
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mso-fareast-font-family:PMingLiU;
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mso-fareast-theme-font:minor-fareast;
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mso-bidi-font-family:Arial;
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mso-bidi-theme-font:minor-bidi;}
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@list l22:level2
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{mso-level-number-format:bullet;
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mso-level-text:o;
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mso-level-number-position:left;
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margin-left:90.0pt;
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text-indent:-18.0pt;
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font-family:"Courier New";}
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@list l22:level3
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{mso-level-number-format:bullet;
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mso-level-number-position:left;
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font-family:Wingdings;}
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@list l22:level4
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{mso-level-number-format:bullet;
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mso-level-text:;
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mso-level-number-position:left;
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margin-left:162.0pt;
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font-family:Symbol;}
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@list l22:level5
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{mso-level-number-format:bullet;
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mso-level-number-position:left;
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font-family:"Courier New";}
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@list l22:level6
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{mso-level-number-format:bullet;
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mso-level-tab-stop:none;
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mso-level-number-position:left;
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margin-left:234.0pt;
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font-family:Wingdings;}
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@list l22:level7
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{mso-level-number-format:bullet;
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mso-level-number-position:left;
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font-family:Symbol;}
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@list l22:level8
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{mso-level-number-format:bullet;
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mso-level-number-position:left;
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font-family:"Courier New";}
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@list l22:level9
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{mso-level-number-format:bullet;
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mso-level-number-position:left;
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mso-list-template-ids:1072331046 134807569 134807577 134807579 134807567 134807577 134807579 134807567 134807577 134807579;}
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@list l23:level1
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{mso-level-text:"%1\)";
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mso-level-tab-stop:none;
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mso-level-number-position:left;
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text-indent:-18.0pt;}
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@list l23:level2
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{mso-level-number-format:alpha-lower;
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mso-level-number-position:left;
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text-indent:-18.0pt;}
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@list l23:level3
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{mso-level-number-format:roman-lower;
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mso-level-tab-stop:none;
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@list l23:level4
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{mso-level-tab-stop:none;
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text-indent:-18.0pt;}
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@list l23:level5
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mso-level-tab-stop:none;
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mso-level-number-position:left;
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text-indent:-18.0pt;}
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@list l23:level6
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{mso-level-number-format:roman-lower;
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mso-level-tab-stop:none;
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mso-level-number-position:right;
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@list l23:level7
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@list l23:level8
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mso-level-tab-stop:none;
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text-indent:-18.0pt;}
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@list l23:level9
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{mso-level-number-format:roman-lower;
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mso-level-tab-stop:none;
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mso-level-number-position:right;
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@list l24:level1
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{mso-level-number-format:bullet;
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mso-level-number-position:left;
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font-family:Symbol;}
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@list l24:level2
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{mso-level-number-format:bullet;
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mso-level-tab-stop:none;
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mso-level-number-position:left;
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text-indent:-18.0pt;
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font-family:"Courier New";
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mso-bidi-font-family:"Times New Roman";}
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@list l24:level3
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{mso-level-number-format:bullet;
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mso-level-text:;
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mso-level-tab-stop:none;
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mso-level-number-position:left;
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font-family:Wingdings;}
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@list l24:level4
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{mso-level-number-format:bullet;
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mso-level-number-position:left;
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font-family:Symbol;}
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@list l24:level5
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{mso-level-number-format:bullet;
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mso-level-tab-stop:none;
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mso-level-number-position:left;
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font-family:"Courier New";
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mso-bidi-font-family:"Times New Roman";}
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@list l24:level6
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{mso-level-number-format:bullet;
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mso-level-tab-stop:none;
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font-family:Wingdings;}
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@list l24:level7
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{mso-level-number-format:bullet;
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mso-level-number-position:left;
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font-family:Symbol;}
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@list l24:level8
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{mso-level-number-format:bullet;
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mso-level-number-position:left;
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font-family:"Courier New";
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mso-bidi-font-family:"Times New Roman";}
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@list l24:level9
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{mso-level-number-format:bullet;
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mso-level-text:;
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mso-level-number-position:left;
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mso-list-template-ids:1499244478 134807569 134807577 134807579 134807567 134807577 134807579 134807567 134807577 134807579;}
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@list l25:level1
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{mso-level-text:"%1\)";
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mso-level-number-position:left;
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text-indent:-18.0pt;}
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{mso-level-number-format:alpha-lower;
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mso-level-tab-stop:none;
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mso-level-number-position:left;
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@list l25:level3
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@list l25:level4
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@list l25:level5
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text-indent:-18.0pt;}
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@list l25:level6
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{mso-level-number-format:roman-lower;
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@list l25:level7
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@list l25:level8
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@list l25:level9
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{mso-level-number-format:roman-lower;
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@list l26:level1
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mso-level-number-format:bullet;
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mso-level-number-position:left;
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font-family:"Calibri",sans-serif;
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mso-fareast-theme-font:minor-fareast;
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mso-bidi-font-family:Arial;
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mso-bidi-theme-font:minor-bidi;}
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@list l26:level2
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mso-level-number-position:left;
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font-family:"Courier New";}
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@list l26:level3
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{mso-level-number-format:bullet;
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@list l26:level4
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{mso-level-number-format:bullet;
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@list l26:level5
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font-family:"Courier New";}
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@list l26:level6
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{mso-level-number-format:bullet;
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@list l27:level1
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{mso-level-text:"%1\)";
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mso-level-number-position:left;
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@list l27:level3
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@list l27:level4
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@list l27:level5
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@list l27:level6
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@list l27:level8
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@list l28:level1
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text-indent:-18.0pt;}
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@list l28:level5
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text-indent:-18.0pt;}
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{mso-level-number-format:roman-lower;
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@list l28:level8
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text-indent:-18.0pt;}
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@list l28:level9
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@list l29:level2
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mso-level-number-position:left;
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text-indent:-18.0pt;
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font-family:"Courier New";
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mso-bidi-font-family:"Times New Roman";}
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@list l29:level3
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@list l29:level4
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 +
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 +
</head>
  
−
<div class="column full_size">
+
<body lang=EN-HK style='tab-interval:36.0pt'>
−
<p>
+
−
<strong>17 July</strong>
+
−
</p>
+
−
<p>
+
−
<strong>Transformation of dCas9 plasmid 2019 kit plate 5 well 22N.</strong>
+
−
</p>
+
−
<p>
+
−
<strong>- Kanamycin Resistance plate</strong>
+
−
</p>
+
−
<p>
+
−
<strong>- to E. Coli Neb 5a  </strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong>19 July</strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong>Picked 6 colonies</strong>
+
−
</p>
+
−
<p>
+
−
<strong>-Grow overnight in LB</strong>
+
−
</p>
+
−
<p>
+
−
<strong>Plasmid Purification (Takara)</strong>
+
−
</p>
+
−
<p>
+
−
<strong>-Colony 1 & 2 did not grow</strong>
+
−
</p>
+
−
<p>
+
−
<strong>-Run agarose gel 100V 30 mins</strong>
+
−
</p>
+
−
<p>
+
−
<strong>-Eluted DNA using 30uL elution buffer</strong>
+
−
</p>
+
−
<p>
+
−
<strong>  -Nanodrop      </strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong>22 July</strong>
+
−
</p>
+
−
<p>
+
−
<strong>Plasmid prep</strong>
+
−
</p>
+
−
<p>
+
−
<strong>Run gel for dcas9</strong>
+
−
</p>
+
−
<p>
+
−
<strong>Resuspend dna</strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong>23 July</strong>
+
−
</p>
+
−
<p>
+
  
−
        <strong>1) Assembling frag1 +2 +3</strong>
+
<div class=WordSection1>
−
</p>
+
−
<p>
+
  
−
    <strong>-RE cut + ligation</strong>
+
<div style='mso-element:para-border-div;border:none;border-right:solid windowtext 1.0pt;
−
</p>
+
mso-border-right-alt:solid windowtext .5pt;padding:0cm 0cm 0cm 0cm'>
−
<p>
+
  
−
    <strong>NheI MliI BssSI-v2</strong>
+
<p class=MsoNormal style='line-height:150%;border:none;mso-border-right-alt:
−
</p>
+
solid windowtext .5pt;padding:0cm;mso-padding-alt:0cm 0cm 0cm 0cm'><b
−
<p>
+
style='mso-bidi-font-weight:normal'><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>17 <span
 +
style='mso-bidi-font-weight:bold'>July</span></span></b><b><span lang=EN-US
 +
style='font-family:Times;mso-bidi-font-family:"Times New Roman";mso-ansi-language:
 +
EN-US'><span style='mso-tab-count:6'>                                                                        </span></span></b><b
 +
style='mso-bidi-font-weight:normal'><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'><o:p></o:p></span></b></p>
  
−
    <strong> </strong>
+
</div>
−
</p>
+
−
<p>
+
  
−
        <strong>2) dcas9 preparation Clean</strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Transformation
−
<p>
+
of dcas9 plasmid 2019 kit plate 5 well 22N<span style='mso-spacerun:yes'>  
 +
</span><o:p></o:p></span></p>
  
−
        <strong>3) Run gel Anson 5α</strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>to E. coli
−
<p>
+
NEB5</span><span lang=EN-US style='font-family:Symbol;mso-fareast-font-family:
 +
Symbol;mso-bidi-font-family:Symbol;mso-ansi-language:EN-US'>a</span><span
 +
lang=EN-US style='font-family:"Times New Roman",serif;mso-fareast-font-family:
 +
"Times New Roman";mso-ansi-language:EN-US'> using kanamycin resistance plate<o:p></o:p></span></p>
  
−
    <strong> </strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'><o:p>&nbsp;</o:p></span></p>
−
<p>
+
  
−
    <strong> </strong>
+
<p class=MsoNormal><b style='mso-bidi-font-weight:normal'><span lang=EN-US
−
</p>
+
style='font-family:"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";
−
<p>
+
mso-ansi-language:EN-US'>19 <span style='mso-bidi-font-weight:bold'>July</span><o:p></o:p></span></b></p>
−
<strong>Transformed produce to Ecoli NEB5a</strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
  
−
        <strong>4) Cloned GFP to dCas9</strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Picked 6
−
<p>
+
colonies<o:p></o:p></span></p>
  
−
    <strong>-RE cut + ligation</strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>grow
−
<p>
+
overnight in LB<o:p></o:p></span></p>
  
−
    <strong>EcoRI NsiI</strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Plasmid
−
<p>
+
purification (Takara)<o:p></o:p></span></p>
  
−
    <strong> </strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>colony 1
−
<p>
+
&amp; 2 did not grow<o:p></o:p></span></p>
  
−
    <strong>Transformed product to Ecoli NEB5a</strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>run agarose gel
−
<p>
+
for 100v 30 mins<o:p></o:p></span></p>
  
−
    <strong> </strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>eluted DNA
−
<p>
+
using 30ul elution buffer<o:p></o:p></span></p>
  
−
        <strong>5) Transformed PUC19 from stock (for cloning sgRNA later)</strong>
+
<p class=MsoNormal style='tab-stops:91.55pt'><span lang=EN-US style='font-family:
−
</p>
+
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
−
<p>
+
EN-US'>nanodrop the eluted DNA</span><span lang=EN-US style='font-family:Times;
−
<strong> </strong>
+
mso-bidi-font-family:"Times New Roman";mso-ansi-language:EN-US'><span
−
</p>
+
style='mso-tab-count:1'>       </span></span><span lang=EN-US style='font-family:
−
<p>
+
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
−
<strong> </strong>
+
EN-US'><o:p></o:p></span></p>
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong>1 Aug</strong>
+
−
</p>
+
−
<p>
+
−
<strong>Cyanobac arrive, cultured in 15x4 mL medium</strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong>2 Aug</strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong>6 Aug</strong>
+
−
</p>
+
−
<p>
+
−
<strong>Colony PCR</strong>
+
−
</p>
+
−
<p>
+
−
<strong>PCR Clean, RE ligation</strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong>7 Aug</strong>
+
−
</p>
+
−
<p>
+
−
<strong>-gel purification after 75V 45min 0.7 gel</strong>
+
−
</p>
+
−
<p>
+
−
<strong>- Gibson assembly</strong>
+
−
</p>
+
−
<p>
+
−
<strong>- Run Gel for Overnigt SgRNA PCR product</strong>
+
−
</p>
+
−
<p>
+
−
<strong>- RE(2hrs incubation)+ligation for SgRNA and pETBlue</strong>
+
−
</p>
+
−
<p>
+
−
<strong>-Transformed 3in1, pETBlue sgRNA and L3 into NEB5a</strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong>8 Aug</strong>
+
−
</p>
+
−
<p>
+
−
<strong>Colony PCR, run gel, pick clone</strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong>9 August</strong>
+
−
</p>
+
−
<p>
+
  
−
        <strong>1) Checked plates for sgRNA pet blue mcyI</strong>
+
<p class=MsoNormal><b style='mso-bidi-font-weight:normal'><span lang=EN-US
−
</p>
+
style='font-family:"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";
−
<p>
+
mso-ansi-language:EN-US'><o:p>&nbsp;</o:p></span></b></p>
  
−
        <strong>sgRNA pet blue mcyI 2</strong>
+
<p class=MsoNormal><b style='mso-bidi-font-weight:normal'><span lang=EN-US
−
</p>
+
style='font-family:"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";
−
<p>
+
mso-ansi-language:EN-US'>22 <span style='mso-bidi-font-weight:bold'>July</span><o:p></o:p></span></b></p>
  
−
    <strong>sgRNA pet blue mcyB 2</strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Plasmid prep
−
<p>
+
<o:p></o:p></span></p>
  
−
    <strong>3 in 1</strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Run gel for dCas9<o:p></o:p></span></p>
−
<p>
+
  
−
    <strong>Gib 2.1</strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Resuspend DNA<o:p></o:p></span></p>
−
<p>
+
  
−
    <strong>Gib 2.2</strong>
+
<p class=MsoNormal><b style='mso-bidi-font-weight:normal'><span lang=EN-US
−
</p>
+
style='font-family:"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";
−
<p>
+
mso-ansi-language:EN-US'><o:p>&nbsp;</o:p></span></b></p>
  
−
    <strong>-All no colony.</strong>
+
<p class=MsoNormal><b style='mso-bidi-font-weight:normal'><span lang=EN-US
−
</p>
+
style='font-family:"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";
−
<p>
+
mso-ansi-language:EN-US'>23 <span style='mso-bidi-font-weight:bold'>July</span><o:p></o:p></span></b></p>
  
−
        <strong>2) Conducted PCR purification of overnight (14h) RE cut of dCas9 GFP</strong>
+
<div style='mso-element:para-border-div;border:none;border-right:solid windowtext 1.0pt;
−
</p>
+
mso-border-right-alt:solid windowtext .5pt;padding:0cm 4.0pt 0cm 0cm'>
−
<p>
+
  
−
    <strong>-50rxn mixture</strong>
+
<p class=MsoNormal style='border:none;mso-border-right-alt:solid windowtext .5pt;
−
</p>
+
padding:0cm;mso-padding-alt:0cm 4.0pt 0cm 0cm'><span lang=EN-US
−
<p>
+
style='font-family:"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";
 +
mso-ansi-language:EN-US'>Assembling Fragment 1+2+3</span><span lang=EN-US
 +
style='font-family:Times;mso-bidi-font-family:"Times New Roman";mso-ansi-language:
 +
EN-US'><o:p></o:p></span></p>
  
−
        <strong>3) Conducted ligation</strong>
+
</div>
−
</p>
+
−
<p>
+
  
−
    <strong>1:3 & 1:5</strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>restriction
−
<p>
+
endonuclease cut + ligation<o:p></o:p></span></p>
  
−
    <strong>Vector 250ng</strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Using <span
−
<p>
+
class=SpellE>NheI</span> and BssSI-v2<o:p></o:p></span></p>
  
−
    <strong>Both 20uL rxn,</strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Dcas9
−
<p>
+
preparation clean</span><span lang=EN-US style='font-family:Times;mso-bidi-font-family:
 +
"Times New Roman";mso-ansi-language:EN-US'><o:p></o:p></span></p>
  
−
    <strong> </strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Run <span
−
<p>
+
class=GramE>gel <span style='mso-spacerun:yes'> </span>dCas</span>9</span><span
 +
style='font-size:11.0pt;font-family:Times;mso-fareast-font-family:"Times New Roman";
 +
mso-bidi-font-family:"Times New Roman";color:#222222'><o:p></o:p></span></p>
  
−
    <strong>-rm temp for 10 min</strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Transformed
−
<p>
+
produce to <span class=GramE>E.coli</span> neb5</span><span lang=EN-US
 +
style='font-family:Symbol;mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:
 +
"Times New Roman";mso-ansi-language:EN-US'>a<o:p></o:p></span></p>
  
−
    <strong>       Heat inactive at 65°C for 10 min</strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Cloned GFP
−
<p>
+
to dcas9</span><span lang=EN-US style='font-family:Times;mso-bidi-font-family:
 +
"Times New Roman";mso-ansi-language:EN-US'><o:p></o:p></span></p>
  
−
    <strong>        On ice 90min</strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>restriction
−
<p>
+
endonuclease cut + ligation<o:p></o:p></span></p>
−
<strong>-Then add ligase again</strong>
+
−
</p>
+
−
<p>
+
−
<strong>        Ligate over weekend.</strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
  
−
        <strong>4) Amplified frag1 + 2 + 3</strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Using <span
−
<p>
+
class=SpellE>EcorI</span> <span class=SpellE>NsiI</span><o:p></o:p></span></p>
  
−
    <strong>-sgRNA I, sgRNA B, GFP</strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Transformed
−
<p>
+
product to <span class=GramE>E.coli</span> neb5</span><span lang=EN-US
 +
style='font-family:Symbol;mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:
 +
"Times New Roman";mso-ansi-language:EN-US'>a<o:p></o:p></span></p>
  
−
        <strong>For Gibson</strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Transformed puc19
−
<p>
+
from stock (for cloning <span class=SpellE>sgrna</span> later)</span><span
 +
lang=EN-US style='font-family:Times;mso-bidi-font-family:"Times New Roman";
 +
mso-ansi-language:EN-US'><o:p></o:p></span></p>
  
−
    <strong>Frag 1 2 3 gfp, 65°C</strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'><o:p>&nbsp;</o:p></span></p>
−
<p>
+
  
−
    <strong>sgRNAs 55C</strong>
+
<p class=MsoNormal><b><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>1 Aug<o:p></o:p></span></b></p>
−
<p>
+
  
−
    <strong> </strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Cyanobacteria
−
<p>
+
arrived, cultured in 15x4 ml medium<o:p></o:p></span></p>
  
−
        <strong>All 50 uL rxn</strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>GFP-dcas9
−
<p>
+
plasmid ligation (all failed)<o:p></o:p></span></p>
  
−
    <strong> </strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'><o:p>&nbsp;</o:p></span></p>
−
<p>
+
  
−
        <strong>5) Weight powder, for SOC (800mL no glucose/ mgXX) (no yellow)</strong>
+
<p class=MsoNormal><b><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>2 Aug<o:p></o:p></span></b></p>
−
<p>
+
  
−
    <strong>-LB agar (have yellow) 1800uL</strong>
+
<p class=MsoNormal style='line-height:107%'><span lang=EN-US style='font-family:
−
</p>
+
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
−
<p>
+
EN-US'>Colony PCR for L2, 3, 4<o:p></o:p></span></p>
  
−
    <strong>Glucose 9g</strong>
+
<p class=MsoNormal style='line-height:107%'><span lang=EN-US style='font-family:
−
</p>
+
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
−
<p>
+
EN-US'>PCR Fragment 1, 2, 3<o:p></o:p></span></p>
  
−
    <strong> </strong>
+
<p class=MsoNormal style='line-height:107%'><span lang=EN-US style='font-family:
−
</p>
+
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
−
<p>
+
EN-US'>Plasmid purification<o:p></o:p></span></p>
  
−
        <strong>6) Run gel for all 6 frags (all in)</strong>
+
<p class=MsoNormal style='line-height:107%'><span lang=EN-US style='font-family:
−
</p>
+
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
−
<p>
+
EN-US'><o:p>&nbsp;</o:p></span></p>
  
−
    <strong>-75V 45 mins</strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>6 Aug<o:p></o:p></span></p>
−
<p>
+
  
−
    <strong>Ladder use 1kb & 50bp</strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>colony PCR <o:p></o:p></span></p>
−
<p>
+
  
−
    <strong>Loading dye 6X</strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>standard q5 PCR
−
<p>
+
<span class=SpellE>preperation</span> for <span class=SpellE>gibson</span> <o:p></o:p></span></p>
  
−
    <strong> </strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Gibson assembly<o:p></o:p></span></p>
−
<p>
+
  
−
        <strong>7) Gel Purification -elude using 30uL elution b.</strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>GFP+ dCas9
−
<p>
+
plasmid again as L3 Failed<o:p></o:p></span></p>
−
<strong>12 August</strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
  
−
        <strong>1) Colony PCR for those without bands reaction</strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'><o:p>&nbsp;</o:p></span></p>
−
<p>
+
  
−
    <strong>-Picked colonies from master plate</strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'><o:p>&nbsp;</o:p></span></p>
−
<p>
+
  
−
    <strong> </strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'><o:p>&nbsp;</o:p></span></p>
−
<p>
+
  
−
        <strong>2) Performed Gibson(Gib3) & NEBuilder</strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'><o:p>&nbsp;</o:p></span></p>
−
<p>
+
  
−
        <strong>-       Scaled down to 10mL rxn</strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>7 Aug<o:p></o:p></span></p>
−
<p>
+
  
−
        <strong>-      Concentration acc to following nanodrop</strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>gel
−
<p>
+
purification after 75v 45min 0.7 gel <o:p></o:p></span></p>
  
−
    <strong>-incubation 1h</strong>
+
<p class=MsoNormal><span class=SpellE><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>gibson</span></span><span
−
<p>
+
lang=EN-US style='font-family:"Times New Roman",serif;mso-fareast-font-family:
−
<strong> </strong>
+
"Times New Roman";mso-ansi-language:EN-US'> assembly <o:p></o:p></span></p>
−
</p>
+
−
<p>
+
  
−
        <strong>3) PCR for XGib 1 (frag1) Gib1 (frag1)</strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>run gel for overnight
−
<p>
+
<span class=SpellE>sgrna</span> PCR product <o:p></o:p></span></p>
−
<strong>                    XGib 2 (frag2) Gib2 (frag2)</strong>
+
−
</p>
+
−
<p>
+
−
<strong>                    XGib 3 (frag3) Gib3 (frag3)</strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
  
−
        <strong>4) Colony PCR no.24 correct band</strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>restriction
−
<p>
+
endonuclease cut (<span class=SpellE>PstI</span>, <span class=SpellE><span
 +
class=GramE>HindIII</span></span><span class=GramE>)(</span>2hrs
 +
incubation)+ligation for sgRNA and <span class=SpellE>PETblue</span> <o:p></o:p></span></p>
  
−
        <strong>(dcas9 gfp) L3</strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>transformed
−
<p>
+
3in1, <span class=SpellE>PETblue</span> sgRNA and L3 into NEB5</span><span
−
<strong>        Band size 700-800 bp</strong>
+
lang=EN-US style='font-family:Symbol;mso-fareast-font-family:Symbol;mso-bidi-font-family:
−
</p>
+
Symbol;mso-ansi-language:EN-US'>a</span><span lang=EN-US style='font-family:
−
<p>
+
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
−
<strong>       Inoculated 24 (picked from master plate)</strong>
+
EN-US'><o:p></o:p></span></p>
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
  
−
        <strong>5) Transformed Gibson 1</strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'><o:p>&nbsp;</o:p></span></p>
−
<p>
+
−
<strong>                          Gibson 2</strong>
+
−
</p>
+
−
<p>
+
−
<strong>        Gibson 3.2 NEBuilder (1)</strong>
+
−
</p>
+
−
<p>
+
−
<strong>        +ve control  petblue NEBuilder (2)</strong>
+
−
</p>
+
−
<p>
+
−
<strong>                          dCas9 gfp 1:3</strong>
+
−
</p>
+
−
<p>
+
−
<strong>                          dCas9 gfp 1:5</strong>
+
−
</p>
+
−
<p>
+
−
<strong>                          Gibson 3.1</strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong>13 August</strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
  
−
        <strong>1) Run gel for previous PCR</strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>8 Aug<o:p></o:p></span></p>
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
  
−
        <strong>2) Checked plates from 12/8</strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Colony PCR
−
<p>
+
for l3 <o:p></o:p></span></p>
  
−
    <strong>Colony: petblue +ve</strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Run gel for
−
<p>
+
3in1 (1 smear), <o:p></o:p></span></p>
  
−
    <strong>       dCas9 gfp 1:3</strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Pick clone<o:p></o:p></span></p>
−
<p>
+
  
−
    <strong>       dCas9 gfp 1:5</strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'><o:p>&nbsp;</o:p></span></p>
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
  
−
        <strong>3) Plasmid purification L3</strong>
+
<p class=MsoNormal><b><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>9 August</span></b><b
−
<p>
+
style='mso-bidi-font-weight:normal'><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'><o:p></o:p></span></b></p>
  
−
    <strong>dCas9 gfp 24</strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Checked
−
<p>
+
plates for sgRNA <span class=SpellE>PETblue</span> <span class=SpellE><span
 +
class=GramE>McyI</span></span><span class=GramE>(</span>no colony)<o:p></o:p></span></p>
  
−
    <strong> </strong>
+
<p class=MsoNormal style='margin-left:72.0pt'><span lang=EN-US
−
</p>
+
style='font-family:Times;mso-bidi-font-family:"Times New Roman";mso-ansi-language:
−
<p>
+
EN-US'><span style='mso-spacerun:yes'>       </span></span><span lang=EN-US
−
<strong>Re Cut for frag1-3 (gib)</strong>
+
style='font-family:"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";
−
</p>
+
mso-ansi-language:EN-US'>sgRNA pet blue <span class=SpellE><span class=GramE>McyI</span></span><span
−
<p>
+
class=GramE>(</span>no colony)<o:p></o:p></span></p>
−
<strong>McyI, McyB</strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong>14 August</strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
  
−
        <strong>1) Run Gel for X gib Frag 1</strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'><span
−
<p>
+
style='mso-spacerun:yes'>                              </span>sgRNA pet blue <span
 +
class=SpellE>Mcyb</span> (no colony)<o:p></o:p></span></p>
  
−
    <strong>-No bands at all</strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'><span
−
<p>
+
style='mso-spacerun:yes'>                              </span>3 in 1(no colony)<o:p></o:p></span></p>
  
−
    <strong>Possibly due to pipetting errors.</strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'><span
−
<p>
+
style='mso-spacerun:yes'>                              </span>Gib 2.1(no
 +
colony)<o:p></o:p></span></p>
  
−
    <strong> </strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'><span
−
<p>
+
style='mso-spacerun:yes'>              </span><span
 +
style='mso-spacerun:yes'>                </span>Gib 2.2(no colony)<o:p></o:p></span></p>
  
−
        <strong>2) Started 10uL reaction using Gibson concentration according to following gel. (pic Gib4)</strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Conducted PCR
−
<p>
+
purification of overnight (14h) restriction endonuclease cut of dCas9 GFP using
 +
<span class=SpellE>EcoRI</span>, <span class=SpellE>NsiI</span></span><span
 +
lang=EN-US style='font-family:Times;mso-bidi-font-family:"Times New Roman";
 +
mso-ansi-language:EN-US'><o:p></o:p></span></p>
  
−
    <strong> </strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>50 </span><span
−
<p>
+
class=SpellE><span style='font-family:Times;mso-fareast-font-family:"Times New Roman";
 +
mso-bidi-font-family:Arial;color:#222222;background:white'>μ<span class=GramE>L</span></span></span><span
 +
class=GramE><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'><span
 +
style='mso-spacerun:yes'>  </span><span class=SpellE>rxn</span></span></span><span
 +
lang=EN-US style='font-family:"Times New Roman",serif;mso-fareast-font-family:
 +
"Times New Roman";mso-ansi-language:EN-US'> mixture </span><span
 +
style='font-family:"Times New Roman",serif;mso-fareast-font-family:"Times New Roman"'><o:p></o:p></span></p>
  
−
    <strong>Reaction: Frag3: 50ng</strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Conducted
−
<p>
+
ligation</span><span lang=EN-US style='font-family:Times;mso-bidi-font-family:
 +
"Times New Roman";mso-ansi-language:EN-US'><o:p></o:p></span></p>
  
−
    <strong>           Frag2: 38.73ng</strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>1:3 &amp;
−
<p>
+
1:5<o:p></o:p></span></p>
  
−
    <strong>           Frag1: 29.26ng</strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Vector 250ng<o:p></o:p></span></p>
−
<p>
+
  
−
    <strong>       Nebuilder: 5uL</strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:Times;mso-fareast-font-family:
−
</p>
+
"Times New Roman";mso-bidi-font-family:"Times New Roman";mso-ansi-language:
−
<p>
+
EN-US'>20 </span><span class=SpellE><span style='font-family:Times;mso-fareast-font-family:
 +
"Times New Roman";mso-bidi-font-family:Arial;color:#222222;background:white'>μL</span></span><span
 +
style='font-family:Times;mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:
 +
Arial;color:#222222;background:white'> <span class=SpellE>rxn</span> mixture</span><span
 +
style='font-family:Times;mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:
 +
"Times New Roman"'><o:p></o:p></span></p>
  
−
    <strong>           H2O</strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Placed in room
−
<p>
+
temperature for 10 min<o:p></o:p></span></p>
  
−
    <strong> </strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>heat
−
<p>
+
inactive at 65</span><span lang=EN-US style='font-family:Symbol;mso-fareast-font-family:
 +
"Times New Roman";mso-bidi-font-family:"Times New Roman";mso-ansi-language:
 +
EN-US'>°</span><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>c for 10 min<o:p></o:p></span></p>
  
−
        <strong>3) Set up PCR reaction for XGib Frag2</strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>on ice 90min<o:p></o:p></span></p>
−
<p>
+
  
−
    <strong>XGib Frag3</strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>then add
−
<p>
+
ligase again<o:p></o:p></span></p>
−
+
−
</p>
+
  
−
<table>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
   <tr>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>ligate over
−
  <td>98°C
+
weekend.<o:p></o:p></span></p>
−
  </td>
+
 
−
  <td>98°C
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
  </td>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Amplified Fragment1
−
  <td>66°
+
+ 2 + 3</span><span lang=EN-US style='font-family:Times;mso-bidi-font-family:
−
  </td>
+
"Times New Roman";mso-ansi-language:EN-US'><o:p></o:p></span></p>
−
  <td>72°C
+
 
−
  </td>
+
<p class=MsoNormal><span class=SpellE><span lang=EN-US style='font-family:"Times New Roman",serif;
−
  <td>72°C
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>sgrna</span></span><span
−
  </td>
+
lang=EN-US style='font-family:"Times New Roman",serif;mso-fareast-font-family:
−
  <td>4°C
+
"Times New Roman";mso-ansi-language:EN-US'> <span class=SpellE>McyI</span>, sgRNA
−
  </td>
+
<span class=SpellE>McyB</span>, GFP<o:p></o:p></span></p>
−
   </tr>
+
 
−
   <tr>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
  <td>30sec
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Fragment 1 2
−
  </td>
+
3 GFP, 65c<o:p></o:p></span></p>
−
  <td>5s
+
 
−
  </td>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
  <td>15s
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>sgRNA 55c<o:p></o:p></span></p>
−
  </td>
+
 
−
  <td>1.5min
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
  </td>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>All 50 </span><span
−
  <td>2min
+
class=SpellE><span style='font-family:"Arial",sans-serif;mso-fareast-font-family:
−
  </td>
+
"Times New Roman";color:#222222;background:white'>μL</span></span><span
−
  <td>¥
+
style='font-family:"Times New Roman",serif;mso-fareast-font-family:"Times New Roman"'>
−
  </td>
+
</span><span class=SpellE><span lang=EN-US style='font-family:"Times New Roman",serif;
−
  </tr>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>rxn</span></span><span
 +
style='font-family:"Times New Roman",serif;mso-fareast-font-family:"Times New Roman"'><o:p></o:p></span></p>
 +
 
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Weight
 +
powder, for soc (800ml no glucose) (no yellow)</span><span lang=EN-US
 +
style='font-family:Times;mso-bidi-font-family:"Times New Roman";mso-ansi-language:
 +
EN-US'><o:p></o:p></span></p>
 +
 
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>LB agar
 +
(have yellow) 1800ul</span><span lang=EN-US style='font-family:Times;
 +
mso-bidi-font-family:"Times New Roman";mso-ansi-language:EN-US'><o:p></o:p></span></p>
 +
 
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Glucose 9g<o:p></o:p></span></p>
 +
 
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Run gel for
 +
all 6 Fragments (all in)</span><span lang=EN-US style='font-family:Times;
 +
mso-bidi-font-family:"Times New Roman";mso-ansi-language:EN-US'><o:p></o:p></span></p>
 +
 
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>75V 45 mins</span><span
 +
lang=EN-US style='font-family:Times;mso-bidi-font-family:"Times New Roman";
 +
mso-ansi-language:EN-US'><o:p></o:p></span></p>
 +
 
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Ladder use
 +
1kb &amp; 50bp<o:p></o:p></span></p>
 +
 
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Loading dye 6x<o:p></o:p></span></p>
 +
 
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Gel purification
 +
-elute using 30ul elution buffer.</span><span lang=EN-US style='font-family:
 +
Times;mso-bidi-font-family:"Times New Roman";mso-ansi-language:EN-US'><o:p></o:p></span></p>
 +
 
 +
<p class=MsoNormal><b><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'><o:p>&nbsp;</o:p></span></b></p>
 +
 
 +
<p class=MsoNormal><b><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'><o:p>&nbsp;</o:p></span></b></p>
 +
 
 +
<p class=MsoNormal><b><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'><o:p>&nbsp;</o:p></span></b></p>
 +
 
 +
<p class=MsoNormal><b><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'><o:p>&nbsp;</o:p></span></b></p>
 +
 
 +
<p class=MsoNormal><b><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'><o:p>&nbsp;</o:p></span></b></p>
 +
 
 +
<p class=MsoNormal><b><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'><o:p>&nbsp;</o:p></span></b></p>
 +
 
 +
<p class=MsoNormal><b style='mso-bidi-font-weight:normal'><span lang=EN-US
 +
style='font-family:"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";
 +
mso-ansi-language:EN-US'>12 <span style='mso-bidi-font-weight:bold'>August</span><o:p></o:p></span></b></p>
 +
 
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Colony PCR
 +
for those without bands reaction </span><span lang=EN-US style='font-family:
 +
Times;mso-bidi-font-family:"Times New Roman";mso-ansi-language:EN-US'><o:p></o:p></span></p>
 +
 
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>picked
 +
colonies from master plate</span><span lang=EN-US style='font-family:Times;
 +
mso-bidi-font-family:"Times New Roman";mso-ansi-language:EN-US'><o:p></o:p></span></p>
 +
 
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Performed <span
 +
class=SpellE>gibson</span>(gib3) &amp; <span class=SpellE>nebuilder</span></span><span
 +
lang=EN-US style='font-family:Times;mso-bidi-font-family:"Times New Roman";
 +
mso-ansi-language:EN-US'><o:p></o:p></span></p>
 +
 
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Scaled down
 +
to 10ml <span class=SpellE>rxn</span></span><span lang=EN-US style='font-family:
 +
Times;mso-bidi-font-family:"Times New Roman";mso-ansi-language:EN-US'><o:p></o:p></span></p>
 +
 
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>PCR for <span
 +
class=SpellE>xgib</span> 1 (Fragment1) gib1 (Fragment1)</span><span lang=EN-US
 +
style='font-family:Times;mso-bidi-font-family:"Times New Roman";mso-ansi-language:
 +
EN-US'><o:p></o:p></span></p>
 +
 
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'><span
 +
style='mso-spacerun:yes'> </span><span style='mso-spacerun:yes'> </span></span><span
 +
lang=EN-US style='font-family:Times;mso-bidi-font-family:"Times New Roman";
 +
mso-ansi-language:EN-US'><span style='mso-tab-count:1'>          </span><span
 +
style='mso-spacerun:yes'> </span><span style='mso-spacerun:yes'> </span></span><span
 +
class=SpellE><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>xgib</span></span><span
 +
lang=EN-US style='font-family:"Times New Roman",serif;mso-fareast-font-family:
 +
"Times New Roman";mso-ansi-language:EN-US'> 2 (Fragment2) gib2 (Fragment2)<o:p></o:p></span></p>
 +
 
 +
<p class=MsoNormal style='text-indent:36.0pt'><span lang=EN-US
 +
style='font-family:"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";
 +
mso-ansi-language:EN-US'><span style='mso-spacerun:yes'> </span><span
 +
style='mso-spacerun:yes'> </span><span class=SpellE>xgib</span> 3 (Fragment3) gib3
 +
(Fragment3)<o:p></o:p></span></p>
 +
 
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'><o:p>&nbsp;</o:p></span></p>
 +
 
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Colony PCR
 +
no.24 correct band</span><span lang=EN-US style='font-family:Times;mso-bidi-font-family:
 +
"Times New Roman";mso-ansi-language:EN-US'><o:p></o:p></span></p>
 +
 
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>(dCas9 GFP) L3</span><span
 +
lang=EN-US style='font-family:Times;mso-bidi-font-family:"Times New Roman";
 +
mso-ansi-language:EN-US'><o:p></o:p></span></p>
 +
 
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>band size
 +
700-800 bp<o:p></o:p></span></p>
 +
 
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>inoculated
 +
24 Hours (picked from master plate)<o:p></o:p></span></p>
 +
 
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'><o:p>&nbsp;</o:p></span></p>
 +
 
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Transformed <span
 +
class=SpellE>gibson</span> 1 <span class=SpellE>gibson</span> 2 <span
 +
class=SpellE>gibson</span> <span class=GramE>3.2<span
 +
style='mso-spacerun:yes'>  </span><span class=SpellE>nebuilder</span></span>
 +
(1)</span><span lang=EN-US style='font-family:Times;mso-bidi-font-family:"Times New Roman";
 +
mso-ansi-language:EN-US'><o:p></o:p></span></p>
 +
 
 +
<p class=MsoNormal><span lang=EN-US style='font-family:Times;mso-bidi-font-family:
 +
"Times New Roman";mso-ansi-language:EN-US'><span style='mso-tab-count:1'>            </span></span><span
 +
lang=EN-US style='font-family:"Times New Roman",serif;mso-fareast-font-family:
 +
"Times New Roman";mso-ansi-language:EN-US'>+<span class=SpellE>ve</span> control<span
 +
style='mso-spacerun:yes'>   </span><span class=SpellE>petblue</span> <span
 +
class=SpellE>nebuilder</span> (2)<o:p></o:p></span></p>
 +
 
 +
<p class=MsoNormal><span lang=EN-US style='font-family:Times;mso-bidi-font-family:
 +
"Times New Roman";mso-ansi-language:EN-US'><span style='mso-tab-count:2'>                        </span></span><span
 +
lang=EN-US style='font-family:"Times New Roman",serif;mso-fareast-font-family:
 +
"Times New Roman";mso-ansi-language:EN-US'><span
 +
style='mso-spacerun:yes'>          </span>dcas9 <span class=SpellE>gfp</span>
 +
1:3<o:p></o:p></span></p>
 +
 
 +
<p class=MsoNormal><span lang=EN-US style='font-family:Times;mso-bidi-font-family:
 +
"Times New Roman";mso-ansi-language:EN-US'><span style='mso-tab-count:2'>                        </span></span><span
 +
lang=EN-US style='font-family:"Times New Roman",serif;mso-fareast-font-family:
 +
"Times New Roman";mso-ansi-language:EN-US'><span
 +
style='mso-spacerun:yes'>          </span>dcas9 <span class=SpellE>gfp</span>
 +
1:5<o:p></o:p></span></p>
 +
 
 +
<p class=MsoNormal><span lang=EN-US style='font-family:Times;mso-bidi-font-family:
 +
"Times New Roman";mso-ansi-language:EN-US'><span style='mso-tab-count:2'>                        </span></span><span
 +
lang=EN-US style='font-family:"Times New Roman",serif;mso-fareast-font-family:
 +
"Times New Roman";mso-ansi-language:EN-US'><span
 +
style='mso-spacerun:yes'>          </span><span class=SpellE>gibson</span> 3.1<o:p></o:p></span></p>
 +
 
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'><o:p>&nbsp;</o:p></span></p>
 +
 
 +
<p class=MsoNormal><b style='mso-bidi-font-weight:normal'><span lang=EN-US
 +
style='font-family:"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";
 +
mso-ansi-language:EN-US'>13 <span style='mso-bidi-font-weight:bold'>August</span><o:p></o:p></span></b></p>
 +
 
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Run gel for
 +
previous PCR </span><span lang=EN-US style='font-family:Times;mso-bidi-font-family:
 +
"Times New Roman";mso-ansi-language:EN-US'><o:p></o:p></span></p>
 +
 
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Checked
 +
plates from 12/8</span><span lang=EN-US style='font-family:Times;mso-bidi-font-family:
 +
"Times New Roman";mso-ansi-language:EN-US'><o:p></o:p></span></p>
 +
 
 +
<p class=MsoNormal style='margin-left:36.0pt'><span lang=EN-US
 +
style='font-family:"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";
 +
mso-ansi-language:EN-US'>Colony: <span class=SpellE>PETblue</span> +<span
 +
class=SpellE>ve</span><o:p></o:p></span></p>
 +
 
 +
<p class=MsoNormal style='margin-left:36.0pt'><span lang=EN-US
 +
style='font-family:Times;mso-bidi-font-family:"Times New Roman";mso-ansi-language:
 +
EN-US'><span style='mso-tab-count:1'>            </span></span><span
 +
lang=EN-US style='font-family:"Times New Roman",serif;mso-fareast-font-family:
 +
"Times New Roman";mso-ansi-language:EN-US'><span
 +
style='mso-spacerun:yes'> </span>dCas9 GFP 1:3<o:p></o:p></span></p>
 +
 
 +
<p class=MsoNormal style='margin-left:36.0pt'><span lang=EN-US
 +
style='font-family:Times;mso-bidi-font-family:"Times New Roman";mso-ansi-language:
 +
EN-US'><span style='mso-tab-count:1'>            </span></span><span
 +
lang=EN-US style='font-family:"Times New Roman",serif;mso-fareast-font-family:
 +
"Times New Roman";mso-ansi-language:EN-US'><span
 +
style='mso-spacerun:yes'> </span>dCas9 GFP 1:5<o:p></o:p></span></p>
 +
 
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Plasmid
 +
purification L3</span><span lang=EN-US style='font-family:Times;mso-bidi-font-family:
 +
"Times New Roman";mso-ansi-language:EN-US'><o:p></o:p></span></p>
 +
 
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>dCas9-GFP 24</span><span
 +
lang=EN-US style='font-family:Times;mso-bidi-font-family:"Times New Roman";
 +
mso-ansi-language:EN-US'><o:p></o:p></span></p>
 +
 
 +
<p class=MsoListParagraph><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'><o:p>&nbsp;</o:p></span></p>
 +
 
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Restriction
 +
endonuclease for Fragment1-3 <span class=GramE>( for</span> <span class=SpellE>gibson</span>
 +
assembly)<o:p></o:p></span></p>
 +
 
 +
<p class=MsoNormal><span class=SpellE><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>McyI</span></span><span
 +
lang=EN-US style='font-family:"Times New Roman",serif;mso-fareast-font-family:
 +
"Times New Roman";mso-ansi-language:EN-US'>, <span class=SpellE>McyB</span><o:p></o:p></span></p>
 +
 
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'><o:p>&nbsp;</o:p></span></p>
 +
 
 +
<p class=MsoNormal><b style='mso-bidi-font-weight:normal'><span lang=EN-US
 +
style='font-family:"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";
 +
mso-ansi-language:EN-US'>14 <span style='mso-bidi-font-weight:bold'>August</span><o:p></o:p></span></b></p>
 +
 
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Run gel for <span
 +
class=SpellE>xgib</span> Fragment 1</span><span lang=EN-US style='font-family:
 +
Times;mso-bidi-font-family:"Times New Roman";mso-ansi-language:EN-US'><o:p></o:p></span></p>
 +
 
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>no bands at
 +
all<o:p></o:p></span></p>
 +
 
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Possibly due
 +
to pipetting errors.<o:p></o:p></span></p>
 +
 
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Started 10ul
 +
reaction using <span class=SpellE>gibson</span> concentration according to
 +
following gel. (pic gib4)</span><span lang=EN-US style='font-family:Times;
 +
mso-bidi-font-family:"Times New Roman";mso-ansi-language:EN-US'><o:p></o:p></span></p>
 +
 
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Reaction: Fragment3:
 +
50ng<o:p></o:p></span></p>
 +
 
 +
<p class=MsoNormal style='margin-left:36.0pt'><span lang=EN-US
 +
style='font-family:"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";
 +
mso-ansi-language:EN-US'><span style='mso-spacerun:yes'>    </span>Fragment2:
 +
38.73ng<o:p></o:p></span></p>
 +
 
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'><span
 +
style='mso-spacerun:yes'>                </span>Fragment1: 29.26ng<o:p></o:p></span></p>
 +
 
 +
<p class=MsoListParagraph><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'><span
 +
style='mso-spacerun:yes'>    </span><span class=SpellE>nebuilder</span>: 5ul<o:p></o:p></span></p>
 +
 
 +
<p class=MsoNormal style='text-indent:18.0pt'><span lang=EN-US
 +
style='font-family:"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";
 +
mso-ansi-language:EN-US'><span style='mso-spacerun:yes'>          </span>h2o<o:p></o:p></span></p>
 +
 
 +
<p class=MsoListParagraphCxSpFirst><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'><o:p>&nbsp;</o:p></span></p>
 +
 
 +
<p class=MsoListParagraphCxSpLast style='text-indent:-18.0pt;mso-list:l27 level1 lfo12'><![if !supportLists]><span
 +
lang=EN-US style='font-family:Times;mso-fareast-font-family:Times;mso-bidi-font-family:
 +
Times;mso-ansi-language:EN-US'><span style='mso-list:Ignore'>1)<span
 +
style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
 +
lang=EN-US style='font-family:"Times New Roman",serif;mso-fareast-font-family:
 +
"Times New Roman";mso-ansi-language:EN-US'>Set up PCR reaction for <span
 +
class=SpellE>xgib</span> Fragment2</span><span lang=EN-US style='font-family:
 +
Times;mso-bidi-font-family:"Times New Roman";mso-ansi-language:EN-US'><o:p></o:p></span></p>
 +
 
 +
<p class=MsoNormal style='margin-left:36.0pt'><span class=SpellE><span
 +
lang=EN-US style='font-family:"Times New Roman",serif;mso-fareast-font-family:
 +
"Times New Roman";mso-ansi-language:EN-US'>Xgib</span></span><span lang=EN-US
 +
style='font-family:"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";
 +
mso-ansi-language:EN-US'> Fragment3<o:p></o:p></span></p>
 +
 
 +
<p class=MsoNormal style='margin-left:36.0pt'><span lang=EN-US
 +
style='font-family:"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";
 +
mso-ansi-language:EN-US'><o:p>&nbsp;</o:p></span></p>
 +
 
 +
<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0
 +
style='margin-left:36.0pt;border-collapse:collapse;border:none;mso-border-alt:
 +
solid windowtext .5pt;mso-yfti-tbllook:1184;mso-padding-alt:0cm 5.4pt 0cm 5.4pt'>
 +
<tr style='mso-yfti-irow:0;mso-yfti-firstrow:yes'>
 +
  <td width=100 valign=top style='width:75.05pt;border:solid windowtext 1.0pt;
 +
  mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt'>
 +
  <p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
  mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>98</span><span
 +
  lang=EN-US style='font-family:Symbol;mso-fareast-font-family:"Times New Roman";
 +
  mso-bidi-font-family:"Times New Roman";mso-ansi-language:EN-US'>°</span><span
 +
  lang=EN-US style='font-family:"Times New Roman",serif;mso-fareast-font-family:
 +
  "Times New Roman";mso-ansi-language:EN-US'>c<o:p></o:p></span></p>
 +
  </td>
 +
  <td width=100 valign=top style='width:75.05pt;border:solid windowtext 1.0pt;
 +
  border-left:none;mso-border-left-alt:solid windowtext .5pt;mso-border-alt:
 +
  solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt'>
 +
  <p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
  mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>98</span><span
 +
  lang=EN-US style='font-family:Symbol;mso-fareast-font-family:"Times New Roman";
 +
  mso-bidi-font-family:"Times New Roman";mso-ansi-language:EN-US'>°</span><span
 +
  lang=EN-US style='font-family:"Times New Roman",serif;mso-fareast-font-family:
 +
  "Times New Roman";mso-ansi-language:EN-US'>c<o:p></o:p></span></p>
 +
  </td>
 +
  <td width=100 valign=top style='width:75.1pt;border:solid windowtext 1.0pt;
 +
  border-left:none;mso-border-left-alt:solid windowtext .5pt;mso-border-alt:
 +
  solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt'>
 +
  <p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
  mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>66</span><span
 +
  lang=EN-US style='font-family:Symbol;mso-fareast-font-family:"Times New Roman";
 +
  mso-bidi-font-family:"Times New Roman";mso-ansi-language:EN-US'>°</span><span
 +
  lang=EN-US style='font-family:"Times New Roman",serif;mso-fareast-font-family:
 +
  "Times New Roman";mso-ansi-language:EN-US'>c<o:p></o:p></span></p>
 +
  </td>
 +
  <td width=100 valign=top style='width:75.1pt;border:solid windowtext 1.0pt;
 +
  border-left:none;mso-border-left-alt:solid windowtext .5pt;mso-border-alt:
 +
  solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt'>
 +
  <p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
  mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>72</span><span
 +
  lang=EN-US style='font-family:Symbol;mso-fareast-font-family:"Times New Roman";
 +
  mso-bidi-font-family:"Times New Roman";mso-ansi-language:EN-US'>°</span><span
 +
  lang=EN-US style='font-family:"Times New Roman",serif;mso-fareast-font-family:
 +
  "Times New Roman";mso-ansi-language:EN-US'>c<o:p></o:p></span></p>
 +
  </td>
 +
  <td width=100 valign=top style='width:75.1pt;border:solid windowtext 1.0pt;
 +
  border-left:none;mso-border-left-alt:solid windowtext .5pt;mso-border-alt:
 +
  solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt'>
 +
  <p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
  mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>72</span><span
 +
  lang=EN-US style='font-family:Symbol;mso-fareast-font-family:"Times New Roman";
 +
  mso-bidi-font-family:"Times New Roman";mso-ansi-language:EN-US'>°</span><span
 +
  lang=EN-US style='font-family:"Times New Roman",serif;mso-fareast-font-family:
 +
  "Times New Roman";mso-ansi-language:EN-US'>c<o:p></o:p></span></p>
 +
  </td>
 +
  <td width=100 valign=top style='width:75.1pt;border:solid windowtext 1.0pt;
 +
  border-left:none;mso-border-left-alt:solid windowtext .5pt;mso-border-alt:
 +
  solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt'>
 +
  <p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
  mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>4</span><span
 +
   lang=EN-US style='font-family:Symbol;mso-fareast-font-family:"Times New Roman";
 +
  mso-bidi-font-family:"Times New Roman";mso-ansi-language:EN-US'>°</span><span
 +
   lang=EN-US style='font-family:"Times New Roman",serif;mso-fareast-font-family:
 +
  "Times New Roman";mso-ansi-language:EN-US'>c<o:p></o:p></span></p>
 +
  </td>
 +
</tr>
 +
<tr style='mso-yfti-irow:1;mso-yfti-lastrow:yes'>
 +
  <td width=100 valign=top style='width:75.05pt;border:solid windowtext 1.0pt;
 +
  border-top:none;mso-border-top-alt:solid windowtext .5pt;mso-border-alt:solid windowtext .5pt;
 +
  padding:0cm 5.4pt 0cm 5.4pt'>
 +
  <p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
  mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>30sec<o:p></o:p></span></p>
 +
  </td>
 +
  <td width=100 valign=top style='width:75.05pt;border-top:none;border-left:
 +
  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
  mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 +
  mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt'>
 +
  <p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
  mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>5sec<o:p></o:p></span></p>
 +
  </td>
 +
  <td width=100 valign=top style='width:75.1pt;border-top:none;border-left:
 +
  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
  mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 +
  mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt'>
 +
  <p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
  mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>15sec<o:p></o:p></span></p>
 +
  </td>
 +
  <td width=100 valign=top style='width:75.1pt;border-top:none;border-left:
 +
  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
  mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 +
  mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt'>
 +
  <p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
  mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>1.5min<o:p></o:p></span></p>
 +
  </td>
 +
  <td width=100 valign=top style='width:75.1pt;border-top:none;border-left:
 +
  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
  mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 +
  mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt'>
 +
  <p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
  mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>2min<o:p></o:p></span></p>
 +
  </td>
 +
  <td width=100 valign=top style='width:75.1pt;border-top:none;border-left:
 +
  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
 +
  mso-border-top-alt:solid windowtext .5pt;mso-border-left-alt:solid windowtext .5pt;
 +
  mso-border-alt:solid windowtext .5pt;padding:0cm 5.4pt 0cm 5.4pt'>
 +
  <p class=MsoNormal><span lang=EN-US style='font-family:Symbol;mso-fareast-font-family:
 +
  "Times New Roman";mso-bidi-font-family:"Times New Roman";mso-ansi-language:
 +
  EN-US'>¥<o:p></o:p></span></p>
 +
  </td>
 +
</tr>
 
</table>
 
</table>
  
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'><o:p>&nbsp;</o:p></span></p>
 +
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'><o:p>&nbsp;</o:p></span></p>
 +
 +
<p class=MsoListParagraphCxSpFirst><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'><o:p>&nbsp;</o:p></span></p>
 +
 +
<p class=MsoListParagraphCxSpMiddle style='text-indent:-18.0pt;mso-list:l27 level1 lfo12'><![if !supportLists]><span
 +
lang=EN-US style='font-family:Times;mso-fareast-font-family:Times;mso-bidi-font-family:
 +
Times;mso-ansi-language:EN-US'><span style='mso-list:Ignore'>2)<span
 +
style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
 +
lang=EN-US style='font-family:"Times New Roman",serif;mso-fareast-font-family:
 +
"Times New Roman";mso-ansi-language:EN-US'>Transformed gib 4.1 gib 4.2</span><span
 +
lang=EN-US style='font-family:Times;mso-bidi-font-family:"Times New Roman";
 +
mso-ansi-language:EN-US'><o:p></o:p></span></p>
 +
 +
<p class=MsoListParagraphCxSpMiddle><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Overnight
 +
ligation <span class=SpellE>sgrna</span> <span class=SpellE>McyI</span> <span
 +
class=SpellE>petblue</span><o:p></o:p></span></p>
 +
 +
<p class=MsoListParagraphCxSpMiddle><span class=SpellE><span lang=EN-US
 +
style='font-family:"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";
 +
mso-ansi-language:EN-US'>Sgrna</span></span><span lang=EN-US style='font-family:
 +
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
 +
EN-US'> <span class=SpellE>Mcyb</span> <span class=SpellE>PETblue</span><o:p></o:p></span></p>
 +
 +
<p class=MsoListParagraphCxSpMiddle><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'><span
 +
style='mso-spacerun:yes'>    </span>gib -0.9ul <span class=SpellE>dna</span></span><span
 +
lang=EN-US style='font-family:Times;mso-bidi-font-family:"Times New Roman";
 +
mso-ansi-language:EN-US'><span style='mso-tab-count:1'>        </span></span><span
 +
lang=EN-US style='font-family:"Times New Roman",serif;mso-fareast-font-family:
 +
"Times New Roman";mso-ansi-language:EN-US'><o:p></o:p></span></p>
 +
 +
<p class=MsoListParagraphCxSpMiddle><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'><span
 +
style='mso-spacerun:yes'>    </span><span class=SpellE>sgrna</span> -2ul<o:p></o:p></span></p>
 +
 +
<p class=MsoListParagraphCxSpMiddle><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'><o:p>&nbsp;</o:p></span></p>
 +
 +
<p class=MsoListParagraphCxSpLast style='text-indent:-18.0pt;mso-list:l27 level1 lfo12'><![if !supportLists]><span
 +
lang=EN-US style='font-family:Times;mso-fareast-font-family:Times;mso-bidi-font-family:
 +
Times;mso-ansi-language:EN-US'><span style='mso-list:Ignore'>3)<span
 +
style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
 +
lang=EN-US style='font-family:"Times New Roman",serif;mso-fareast-font-family:
 +
"Times New Roman";mso-ansi-language:EN-US'>Checked plates for previous
 +
transformation: gib 3 in 1 -</span><span lang=EN-US style='font-family:Times;
 +
mso-bidi-font-family:"Times New Roman";mso-ansi-language:EN-US'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='margin-left:18.0pt'><span lang=EN-US
 +
style='font-family:"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";
 +
mso-ansi-language:EN-US'><span style='mso-spacerun:yes'> </span></span><span
 +
lang=EN-US style='font-family:Times;mso-bidi-font-family:"Times New Roman";
 +
mso-ansi-language:EN-US'><span style='mso-tab-count:7'>                                                                            </span><span
 +
style='mso-spacerun:yes'>      </span></span><span lang=EN-US style='font-family:
 +
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
 +
EN-US'><span style='mso-spacerun:yes'> </span>gib2 - no <span class=SpellE>colonierom</span>
 +
last year -<span class=SpellE>PETblue</span> stock - there are colonies<o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='margin-left:18.0pt'><span lang=EN-US
 +
style='font-family:"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";
 +
mso-ansi-language:EN-US'><o:p>&nbsp;</o:p></span></p>
 +
 +
<p class=MsoListParagraph style='text-indent:-18.0pt;mso-list:l27 level1 lfo12'><![if !supportLists]><span
 +
lang=EN-US style='font-family:Times;mso-fareast-font-family:Times;mso-bidi-font-family:
 +
Times;mso-ansi-language:EN-US'><span style='mso-list:Ignore'>4)<span
 +
style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
 +
lang=EN-US style='font-family:"Times New Roman",serif;mso-fareast-font-family:
 +
"Times New Roman";mso-ansi-language:EN-US'>Picked 6 colonies from <span
 +
class=SpellE>PETblue</span></span><span lang=EN-US style='font-family:Times;
 +
mso-bidi-font-family:"Times New Roman";mso-ansi-language:EN-US'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='margin-left:18.0pt'><span lang=EN-US
 +
style='font-family:Times;mso-bidi-font-family:"Times New Roman";mso-ansi-language:
 +
EN-US'><span style='mso-tab-count:2'>                  </span></span><span
 +
class=SpellE><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>PETblue</span></span><span
 +
lang=EN-US style='font-family:"Times New Roman",serif;mso-fareast-font-family:
 +
"Times New Roman";mso-ansi-language:EN-US'> 1-3 -3ml</span><span lang=EN-US
 +
style='font-family:Times;mso-bidi-font-family:"Times New Roman";mso-ansi-language:
 +
EN-US'> </span><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>(with amp)<o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='margin-left:18.0pt'><span lang=EN-US
 +
style='font-family:Times;mso-bidi-font-family:"Times New Roman";mso-ansi-language:
 +
EN-US'><span style='mso-tab-count:2'>                  </span></span><span
 +
class=SpellE><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>PETblue</span></span><span
 +
lang=EN-US style='font-family:"Times New Roman",serif;mso-fareast-font-family:
 +
"Times New Roman";mso-ansi-language:EN-US'> 4-5 -2ml<span
 +
style='mso-spacerun:yes'>      </span><span style='mso-spacerun:yes'>  </span><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='margin-left:18.0pt'><span lang=EN-US
 +
style='font-family:"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";
 +
mso-ansi-language:EN-US'>Master plate -no <span class=SpellE>xgal</span> <span
 +
class=SpellE>iptg</span><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='margin-left:18.0pt'><span lang=EN-US
 +
style='font-family:"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";
 +
mso-ansi-language:EN-US'><o:p>&nbsp;</o:p></span></p>
 +
 +
<p class=MsoListParagraph style='text-indent:-18.0pt;mso-list:l27 level1 lfo12'><![if !supportLists]><span
 +
lang=EN-US style='font-family:Times;mso-fareast-font-family:Times;mso-bidi-font-family:
 +
Times;mso-ansi-language:EN-US'><span style='mso-list:Ignore'>5)<span
 +
style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp; </span></span></span><![endif]><span
 +
lang=EN-US style='font-family:"Times New Roman",serif;mso-fareast-font-family:
 +
"Times New Roman";mso-ansi-language:EN-US'>Run gel for PCR products </span><span
 +
lang=EN-US style='font-family:Times;mso-bidi-font-family:"Times New Roman";
 +
mso-ansi-language:EN-US'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='margin-left:36.0pt'><span lang=EN-US
 +
style='font-family:"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";
 +
mso-ansi-language:EN-US'>Fragment 2, Fragment 3 <span class=SpellE>xgib</span><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='margin-left:36.0pt'><span lang=EN-US
 +
style='font-family:"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";
 +
mso-ansi-language:EN-US'>Gel purification<o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='margin-left:36.0pt'><span lang=EN-US
 +
style='font-family:"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";
 +
mso-ansi-language:EN-US'>DNA concentration on 13/8<o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><b style='mso-bidi-font-weight:normal'><span lang=EN-US
 +
style='font-family:"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";
 +
mso-ansi-language:EN-US'><o:p>&nbsp;</o:p></span></b></p>
 +
 +
<p class=MsoNormal><b style='mso-bidi-font-weight:normal'><span lang=EN-US
 +
style='font-family:"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";
 +
mso-ansi-language:EN-US'>15 <span style='mso-bidi-font-weight:bold'>August</span>
 +
<o:p></o:p></span></b></p>
 +
 +
<p class=MsoNormal><b style='mso-bidi-font-weight:normal'><span lang=EN-US
 +
style='font-family:"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";
 +
mso-ansi-language:EN-US'><o:p>&nbsp;</o:p></span></b></p>
 +
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Restriction
 +
endonuclease cut for Fragment1 Fragment 2 and Fragment 3</span><span
 +
lang=EN-US style='font-family:Times;mso-bidi-font-family:"Times New Roman";
 +
mso-ansi-language:EN-US'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span class=SpellE><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Xgib</span></span><span
 +
lang=EN-US style='font-family:"Times New Roman",serif;mso-fareast-font-family:
 +
"Times New Roman";mso-ansi-language:EN-US'> ~6 <span class=SpellE>hr</span> (<span
 +
class=SpellE>MluI</span>, <span class=SpellE>BssaI</span> and <span
 +
class=SpellE>NheI</span>)<o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>nanodrop
 +
before restriction endonuclease cut<o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Set up <span
 +
class=SpellE>gibson</span> reaction</span><span lang=EN-US style='font-family:
 +
Times;mso-bidi-font-family:"Times New Roman";mso-ansi-language:EN-US'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='text-indent:36.0pt'><span lang=EN-US
 +
style='font-family:"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";
 +
mso-ansi-language:EN-US'>Fragment 3 50ng<span style='mso-spacerun:yes'>   
 +
</span><span style='mso-spacerun:yes'> </span>16ul</span><span lang=EN-US
 +
style='font-family:Times;mso-bidi-font-family:"Times New Roman";mso-ansi-language:
 +
EN-US'><span style='mso-tab-count:2'>                    </span><span
 +
style='mso-tab-count:1'>            </span><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='text-indent:36.0pt'><span lang=EN-US
 +
style='font-family:"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";
 +
mso-ansi-language:EN-US'>Fragment 2 38.73ng 2.1 ul</span><span lang=EN-US
 +
style='font-family:Times;mso-bidi-font-family:"Times New Roman";mso-ansi-language:
 +
EN-US'><span style='mso-tab-count:2'>                  </span><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='text-indent:36.0pt'><span lang=EN-US
 +
style='font-family:"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";
 +
mso-ansi-language:EN-US'>Fragment 1 29.26ng 0.4ul</span><span lang=EN-US
 +
style='font-family:Times;mso-bidi-font-family:"Times New Roman";mso-ansi-language:
 +
EN-US'><span style='mso-tab-count:2'>                  </span><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='text-indent:36.0pt'><span class=SpellE><span
 +
lang=EN-US style='font-family:"Times New Roman",serif;mso-fareast-font-family:
 +
"Times New Roman";mso-ansi-language:EN-US'>Nebuilder</span></span><span
 +
lang=EN-US style='font-family:"Times New Roman",serif;mso-fareast-font-family:
 +
"Times New Roman";mso-ansi-language:EN-US'><span
 +
style='mso-spacerun:yes'>          </span><span
 +
style='mso-spacerun:yes'>      </span>10ul</span><span lang=EN-US
 +
style='font-family:Times;mso-bidi-font-family:"Times New Roman";mso-ansi-language:
 +
EN-US'><span style='mso-tab-count:2'>                    </span><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='text-indent:36.0pt'><span lang=EN-US
 +
style='font-family:"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";
 +
mso-ansi-language:EN-US'>H2o</span><span lang=EN-US style='font-family:Times;
 +
mso-bidi-font-family:"Times New Roman";mso-ansi-language:EN-US'><span
 +
style='mso-tab-count:2'>                </span></span><span lang=EN-US
 +
style='font-family:"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";
 +
mso-ansi-language:EN-US'><span style='mso-spacerun:yes'>        </span>5.9ul</span><span
 +
lang=EN-US style='font-family:Times;mso-bidi-font-family:"Times New Roman";
 +
mso-ansi-language:EN-US'><span style='mso-tab-count:2'>                  </span></span><span
 +
lang=EN-US style='font-family:"Times New Roman",serif;mso-fareast-font-family:
 +
"Times New Roman";mso-ansi-language:EN-US'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='text-indent:36.0pt'><span lang=EN-US
 +
style='font-family:"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";
 +
mso-ansi-language:EN-US'>50</span><span lang=EN-US style='font-family:Symbol;
 +
mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:"Times New Roman";
 +
mso-ansi-language:EN-US'>°</span><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>c for 2hr
 +
(optimized protocol)<o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'><o:p>&nbsp;</o:p></span></p>
 +
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Plasmid purification
 +
of <span class=SpellE>PETblue</span> </span><span lang=EN-US style='font-family:
 +
Times;mso-bidi-font-family:"Times New Roman";mso-ansi-language:EN-US'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'><span
 +
style='mso-spacerun:yes'> </span><span style='mso-spacerun:yes'>      </span><span
 +
style='mso-spacerun:yes'>    </span>eluted using 30ul elution buffer<o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span lang=EN-US style='font-family:Times;mso-bidi-font-family:
 +
"Times New Roman";mso-ansi-language:EN-US'><span style='mso-tab-count:1'>            </span></span><span
 +
lang=EN-US style='font-family:"Times New Roman",serif;mso-fareast-font-family:
 +
"Times New Roman";mso-ansi-language:EN-US'>nanodrop<o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'><o:p>&nbsp;</o:p></span></p>
 +
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Gib 4.1 -<span
 +
class=SpellE>PETblue</span> <span class=SpellE>McyB</span> plasmid have 1
 +
colony each</span><span lang=EN-US style='font-family:Times;mso-bidi-font-family:
 +
"Times New Roman";mso-ansi-language:EN-US'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'><span
 +
style='mso-spacerun:yes'> </span></span><span lang=EN-US style='font-family:
 +
Times;mso-bidi-font-family:"Times New Roman";mso-ansi-language:EN-US'><span
 +
style='mso-tab-count:1'>          </span><span
 +
style='mso-spacerun:yes'> </span></span><span lang=EN-US style='font-family:
 +
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
 +
EN-US'>picked &amp; inoculated<o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'><o:p>&nbsp;</o:p></span></p>
 +
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'><o:p>&nbsp;</o:p></span></p>
 +
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>PCR purified
 +
restriction endonuclease cuts from (Fragment 1, 2, 3)</span><span lang=EN-US
 +
style='font-family:Times;mso-bidi-font-family:"Times New Roman";mso-ansi-language:
 +
EN-US'><o:p></o:p></span></p>
 +
 +
<p class=MsoListParagraphCxSpFirst><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>ligated h3
 +
&amp; 3 in 1<o:p></o:p></span></p>
 +
 +
<p class=MsoListParagraphCxSpMiddle><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>1+3 </span><span
 +
lang=EN-US style='font-family:Times;mso-bidi-font-family:"Times New Roman";
 +
mso-ansi-language:EN-US'><span style='mso-tab-count:1'>    </span></span><span
 +
lang=EN-US style='font-family:"Times New Roman",serif;mso-fareast-font-family:
 +
"Times New Roman";mso-ansi-language:EN-US'>1:1<o:p></o:p></span></p>
 +
 +
<p class=MsoListParagraphCxSpLast><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>1+2+3</span><span
 +
lang=EN-US style='font-family:Times;mso-bidi-font-family:"Times New Roman";
 +
mso-ansi-language:EN-US'><span style='mso-tab-count:1'> </span></span><span
 +
lang=EN-US style='font-family:"Times New Roman",serif;mso-fareast-font-family:
 +
"Times New Roman";mso-ansi-language:EN-US'>1:1:1<o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Sequencing</span><span
 +
lang=EN-US style='font-family:Times;mso-bidi-font-family:"Times New Roman";
 +
mso-ansi-language:EN-US'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'><o:p>&nbsp;</o:p></span></p>
 +
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'><o:p>&nbsp;</o:p></span></p>
 +
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>16 August<o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'><o:p>&nbsp;</o:p></span></p>
 +
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Plasmid
 +
purification of </span><span lang=EN-US style='font-family:Times;mso-bidi-font-family:
 +
"Times New Roman";mso-ansi-language:EN-US'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Gib4.1 &amp;
 +
pet blue <span class=SpellE>McyB</span> plasmid from 1518<o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Gib4.1 does
 +
not seem to grow during inoculation therefore, low yield.<o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Run gel for Fragment
 +
1+3 &amp; 3in1 (ligated)</span><span lang=EN-US style='font-family:Times;
 +
mso-bidi-font-family:"Times New Roman";mso-ansi-language:EN-US'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Intended to
 +
amp 1+3 using 3f + 1r and 3r + 1f</span><span lang=EN-US style='font-family:
 +
Times;mso-bidi-font-family:"Times New Roman";mso-ansi-language:EN-US'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Intended to check
 +
gib4 using 1+2, 2+3, 1+3 primers.</span><span lang=EN-US style='font-family:
 +
Times;mso-bidi-font-family:"Times New Roman";mso-ansi-language:EN-US'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>3 and 4
 +
failed possibly due to PCR errors<o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>use 3 mins
 +
extension </span><span lang=EN-US style='font-family:Times;mso-bidi-font-family:
 +
"Times New Roman";mso-ansi-language:EN-US'><span style='mso-tab-count:1'>  </span></span><span
 +
lang=EN-US style='font-family:"Times New Roman",serif;mso-fareast-font-family:
 +
"Times New Roman";mso-ansi-language:EN-US'>65</span><span lang=EN-US
 +
style='font-family:Symbol;mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:
 +
"Times New Roman";mso-ansi-language:EN-US'>°</span><span lang=EN-US
 +
style='font-family:"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";
 +
mso-ansi-language:EN-US'>c </span><span lang=EN-US style='font-family:Times;
 +
mso-bidi-font-family:"Times New Roman";mso-ansi-language:EN-US'><span
 +
style='mso-tab-count:1'>    </span></span><span lang=EN-US style='font-family:
 +
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
 +
EN-US'>other times and maximum<o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Intended to
 +
check sgRNA <span class=SpellE>Mcyb</span> <span class=SpellE>PETblue</span>
 +
using colony PCR</span><span lang=EN-US style='font-family:Times;mso-bidi-font-family:
 +
"Times New Roman";mso-ansi-language:EN-US'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>(failed due
 +
to 0.7% gel)<o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><b style='mso-bidi-font-weight:normal'><span lang=EN-US
 +
style='font-family:"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";
 +
mso-ansi-language:EN-US'><o:p>&nbsp;</o:p></span></b></p>
 +
 +
<p class=MsoNormal><b style='mso-bidi-font-weight:normal'><span lang=EN-US
 +
style='font-family:"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";
 +
mso-ansi-language:EN-US'>19 <span style='mso-bidi-font-weight:bold'>August</span>
 +
<o:p></o:p></span></b></p>
 +
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Colony PCR
 +
for <span class=SpellE>Mcyb</span> sgRNA <span class=SpellE>PETblue</span></span><span
 +
lang=EN-US style='font-family:Times;mso-bidi-font-family:"Times New Roman";
 +
mso-ansi-language:EN-US'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>(only 1
 +
white colony)</span><span lang=EN-US style='font-family:Times;mso-bidi-font-family:
 +
"Times New Roman";mso-ansi-language:EN-US'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Results
 +
positive: band size ~300bp<o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Inoculated 2
 +
3ml tubes of gib4</span><span lang=EN-US style='font-family:Times;mso-bidi-font-family:
 +
"Times New Roman";mso-ansi-language:EN-US'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>gib4.1 &amp;
 +
4.2</span><span lang=EN-US style='font-family:Times;mso-bidi-font-family:"Times New Roman";
 +
mso-ansi-language:EN-US'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>From master
 +
plate</span><span lang=EN-US style='font-family:Times;mso-bidi-font-family:
 +
"Times New Roman";mso-ansi-language:EN-US'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Set up
 +
overnight PCR to amp <span class=SpellE>mcyI</span>, Fragment1, Fragment3 <span
 +
class=SpellE>xgib</span>,</span><span lang=EN-US style='font-family:Times;
 +
mso-bidi-font-family:"Times New Roman";mso-ansi-language:EN-US'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Restriction
 +
endonuclease (<span class=SpellE>PstI</span>, <span class=SpellE>HindIII</span>,
 +
<span class=SpellE>MluI</span>, <span class=SpellE>BssaI</span>, <span
 +
class=SpellE>NheI</span>) cut <span class=SpellE>McyI</span> (247.8ng), <span
 +
class=SpellE>PETblue</span> (1000ng), Fragment1 (741ng)- Fragment3 (140ng)
 +
overnight [<span class=SpellE>McyI</span> only added 3ul]</span><span
 +
lang=EN-US style='font-family:Times;mso-bidi-font-family:"Times New Roman";
 +
mso-ansi-language:EN-US'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Checked neb
 +
3 in 1 using different primers </span><span lang=EN-US style='font-family:Times;
 +
mso-bidi-font-family:"Times New Roman";mso-ansi-language:EN-US'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Checked Fragment
 +
1+3<o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'><o:p>&nbsp;</o:p></span></p>
 +
 +
<p class=MsoNormal><b style='mso-bidi-font-weight:normal'><span lang=EN-US
 +
style='font-family:"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";
 +
mso-ansi-language:EN-US'><span style='mso-spacerun:yes'> </span>20 <span
 +
style='mso-bidi-font-weight:bold'>August</span> <o:p></o:p></span></b></p>
 +
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Run gel for <span
 +
class=SpellE>mcyI</span>, Fragment1-3 <span class=SpellE>xgib</span></span><span
 +
lang=EN-US style='font-family:Times;mso-bidi-font-family:"Times New Roman";
 +
mso-ansi-language:EN-US'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>gel purification
 +
Takara<o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>PCR clean <span
 +
class=SpellE>McyI</span>, <span class=SpellE>PETblue</span> 2 from overnight restriction
 +
endonuclease cut</span><span lang=EN-US style='font-family:Times;mso-bidi-font-family:
 +
"Times New Roman";mso-ansi-language:EN-US'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Ligated <span
 +
class=SpellE>McyI</span> <span class=SpellE>PETblue</span>, Fragment1+3</span><span
 +
lang=EN-US style='font-family:Times;mso-bidi-font-family:"Times New Roman";
 +
mso-ansi-language:EN-US'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>vector 100ng</span><span
 +
lang=EN-US style='font-family:Times;mso-bidi-font-family:"Times New Roman";
 +
mso-ansi-language:EN-US'><span style='mso-tab-count:1'>  </span></span><span
 +
lang=EN-US style='font-family:"Times New Roman",serif;mso-fareast-font-family:
 +
"Times New Roman";mso-ansi-language:EN-US'>-vector 100ng<o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>1:3</span><span
 +
lang=EN-US style='font-family:Times;mso-bidi-font-family:"Times New Roman";
 +
mso-ansi-language:EN-US'><span style='mso-tab-count:3'>                              </span></span><span
 +
lang=EN-US style='font-family:"Times New Roman",serif;mso-fareast-font-family:
 +
"Times New Roman";mso-ansi-language:EN-US'><span style='mso-spacerun:yes'> 
 +
</span>1:1<o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Plasmid purification
 +
gib6.4</span><span lang=EN-US style='font-family:Times;mso-bidi-font-family:
 +
"Times New Roman";mso-ansi-language:EN-US'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>yield
 +
extremely low<o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Picked
 +
colonies: 2 from <span class=SpellE>nebuilder</span> 3in1.</span><span
 +
lang=EN-US style='font-family:Times;mso-bidi-font-family:"Times New Roman";
 +
mso-ansi-language:EN-US'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='margin-left:72.0pt'><span lang=EN-US
 +
style='font-family:"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";
 +
mso-ansi-language:EN-US'><span style='mso-spacerun:yes'> </span><span
 +
style='mso-spacerun:yes'>  </span><span style='mso-spacerun:yes'> </span>2 from
 +
gib<span class=GramE>4<span style='mso-spacerun:yes'>  </span>-</span>master
 +
plate<o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='margin-left:72.0pt'><span lang=EN-US
 +
style='font-family:"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";
 +
mso-ansi-language:EN-US'><span style='mso-spacerun:yes'>    </span>2 from <span
 +
class=SpellE>sgrRNA</span> <span class=SpellE>McyB</span> <span class=SpellE>PETblue</span>-<o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><b style='mso-bidi-font-weight:normal'><span lang=EN-US
 +
style='font-family:"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";
 +
mso-ansi-language:EN-US'><o:p>&nbsp;</o:p></span></b></p>
 +
 +
<p class=MsoNormal><b style='mso-bidi-font-weight:normal'><span lang=EN-US
 +
style='font-family:"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";
 +
mso-ansi-language:EN-US'>21 <span style='mso-bidi-font-weight:bold'>August</span><o:p></o:p></span></b></p>
 +
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Plasmid <span
 +
class=SpellE>purifucation</span> for 6 tubes - <span class=SpellE>nebuilder</span>
 +
x2, gib4 x2, <span class=SpellE>Sgrna</span> <span class=SpellE>Mcyb</span> <span
 +
class=SpellE>PETblue</span></span><span lang=EN-US style='font-family:Times;
 +
mso-bidi-font-family:"Times New Roman";mso-ansi-language:EN-US'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Collected
 +
overnight ligation</span><span lang=EN-US style='font-family:Times;mso-bidi-font-family:
 +
"Times New Roman";mso-ansi-language:EN-US'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>amplified Fragment
 +
1+3 <o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Primer Fragment1
 +
f &amp; primer Fragment3 (vice versa also)<o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Transformed sgRNA
 +
<span class=SpellE>McyI</span> <span class=SpellE>PETblue</span> and dCas9 GFP
 +
(L3)</span><span lang=EN-US style='font-family:Times;mso-bidi-font-family:"Times New Roman";
 +
mso-ansi-language:EN-US'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Received
 +
sanger sequence results </span><span lang=EN-US style='font-family:Times;
 +
mso-bidi-font-family:"Times New Roman";mso-ansi-language:EN-US'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>99.4%
 +
identical</span><span lang=EN-US style='font-family:Times;mso-bidi-font-family:
 +
"Times New Roman";mso-ansi-language:EN-US'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Mutation <o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span lang=EN-US style='font-family:Symbol;mso-fareast-font-family:
 +
"Times New Roman";mso-bidi-font-family:"Times New Roman";mso-ansi-language:
 +
EN-US'>\</span><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'> wrong</span><span
 +
lang=EN-US style='font-family:Times;mso-bidi-font-family:"Times New Roman";
 +
mso-ansi-language:EN-US'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'><o:p>&nbsp;</o:p></span></p>
 +
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Picked 25
 +
colonies from previous l3 plate</span><span lang=EN-US style='font-family:Times;
 +
mso-bidi-font-family:"Times New Roman";mso-ansi-language:EN-US'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>colony PCR </span><span
 +
lang=EN-US style='font-family:Times;mso-bidi-font-family:"Times New Roman";
 +
mso-ansi-language:EN-US'><span style='mso-tab-count:2'>                </span></span><span
 +
lang=EN-US style='font-family:"Times New Roman",serif;mso-fareast-font-family:
 +
"Times New Roman";mso-ansi-language:EN-US'>25-49<span
 +
style='mso-spacerun:yes'>  </span>-One <span class=SpellE>Taq</span><o:p></o:p></span></p>
 +
 +
<p class=MsoListParagraph><span lang=EN-US style='font-family:Times;mso-bidi-font-family:
 +
"Times New Roman";mso-ansi-language:EN-US'><span style='mso-tab-count:2'>                        </span></span><span
 +
lang=EN-US style='font-family:"Times New Roman",serif;mso-fareast-font-family:
 +
"Times New Roman";mso-ansi-language:EN-US'>50</span><span lang=EN-US
 +
style='font-family:Times;mso-bidi-font-family:"Times New Roman";mso-ansi-language:
 +
EN-US'><span style='mso-tab-count:1'>        </span></span><span lang=EN-US
 +
style='font-family:"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";
 +
mso-ansi-language:EN-US'>- Sapphire<o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Single cut
 +
&amp; double cut 3 in 1 plasmid</span><span lang=EN-US style='font-family:Times;
 +
mso-bidi-font-family:"Times New Roman";mso-ansi-language:EN-US'><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>all
 +
undesired results<o:p></o:p></span></p>
 +
 +
<p class=MsoListParagraph><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'><o:p>&nbsp;</o:p></span></p>
 +
 +
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
 +
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
 +
EN-US'><span style='mso-spacerun:yes'> </span>22 Aug<o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
 +
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
 +
EN-US'><span style='mso-spacerun:yes'> </span>Gibson assembly, incubate 2h<o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
 +
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
 +
EN-US'><span style='mso-spacerun:yes'> </span>run gel for colony PCR and Fragment
 +
1+3 and the overnight cut of 3 in 1<o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
 +
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
 +
EN-US'>For colonies with correct band after colony PCR, pick colony from master
 +
plate, inoculate<o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
 +
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
 +
EN-US'>Fragment 1+3, Fragment 2 re cut, <o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
 +
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
 +
EN-US'>Send <span class=SpellE>McyB</span> to <span class=GramE>sequencing</span><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
 +
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
 +
EN-US'>Transform <span class=SpellE>gibson</span> products<o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
 +
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
 +
EN-US'>Ligate Fragment 2 to 1+3<o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
 +
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
 +
EN-US'><o:p>&nbsp;</o:p></span></p>
 +
 +
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
 +
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
 +
EN-US'>23 Aug<o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
 +
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
 +
EN-US'>Run gel for PCR dCas9 GFP, <o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
 +
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
 +
EN-US'>Run gel for PCR 1+3 sgRNA MCYI <span class=SpellE>PETblue</span><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
 +
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
 +
EN-US'><o:p>&nbsp;</o:p></span></p>
 +
 +
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
 +
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
 +
EN-US'>26 Aug<o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
 +
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
 +
EN-US'>Run gel for previous 40 colony PCR<o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
 +
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
 +
EN-US'>Inoculate l3 with correct band size<o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
 +
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
 +
EN-US'>Preparation for electroporation<o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
 +
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
 +
EN-US'>Transform ligation product and 3 in 1 for characterization<o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
 +
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
 +
EN-US'><o:p>&nbsp;</o:p></span></p>
 +
 +
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
 +
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
 +
EN-US'>27 Aug<o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
 +
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
 +
EN-US'>Pick clone for previous transformation<o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
 +
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
 +
EN-US'>3 in 1 plasmid purification using ethanol perpetration <o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
 +
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
 +
EN-US'>Restriction endonuclease cut gib 5 (<span class=SpellE>MluI</span>)<o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
 +
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
 +
EN-US'><o:p>&nbsp;</o:p></span></p>
 +
 +
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
 +
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
 +
EN-US'>28 Aug<o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
 +
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
 +
EN-US'>PCR screening <span class=SpellE>McyI</span>, <span class=SpellE>McyB</span><o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
 +
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
 +
EN-US'><o:p>&nbsp;</o:p></span></p>
 +
 +
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
 +
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
 +
EN-US'>29 Aug<o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
 +
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
 +
EN-US'>Plasmid purification. Using Takara<o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
 +
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
 +
EN-US'>Inoculate gib 5, prepare for measurement<o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
 +
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";color:red;
 +
mso-ansi-language:EN-US'><o:p>&nbsp;</o:p></span></p>
 +
 +
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
 +
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
 +
EN-US'>3/9<o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
 +
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
 +
EN-US'>Restriction endonuclease cut dCas9 GFP (<span class=SpellE>EcoRI</span>,
 +
<span class=SpellE>PstI</span>)<o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
 +
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
 +
EN-US'><o:p>&nbsp;</o:p></span></p>
 +
 +
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
 +
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
 +
EN-US'>4/9<o:p></o:p></span></p>
 +
 +
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
 +
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Run gel for restriction
 +
endonuclease cut dCas9 GFP<o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
 +
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
 +
EN-US'><o:p>&nbsp;</o:p></span></p>
 +
 +
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
 +
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
 +
EN-US'>5/9<o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
 +
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
 +
EN-US'>Transformation inoculate <span class=SpellE>PETblue</span>, Puc19, 3 in
 +
1, PsB1c3 transformants<o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
 +
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
 +
EN-US'>Plasmid purification 3 in 1 using Takara plasmid purification kit<o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
 +
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
 +
EN-US'><o:p>&nbsp;</o:p></span></p>
 +
 +
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
 +
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
 +
EN-US'>6/9<o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
 +
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
 +
EN-US'>Plasmid purification 3 in 1 using Takara plasmid purification kit<o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
 +
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
 +
EN-US'><o:p>&nbsp;</o:p></span></p>
 +
 +
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
 +
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
 +
EN-US'>9/9<o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
 +
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
 +
EN-US'>2 restriction endonuclease cuts (dCas9-GFP-sgRNA)<o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
 +
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
 +
EN-US'><o:p>&nbsp;</o:p></span></p>
 +
 +
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
 +
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
 +
EN-US'>10/9<o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
 +
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
 +
EN-US'>Run gel for the colony PCR products (1.5 gel, all in) 65v 45 min 700 or
 +
no band<o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
 +
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
 +
EN-US'>Pick colony and colony PCR <o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
 +
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
 +
EN-US'>Pour agar plate <o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
 +
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
 +
EN-US'><span style='mso-spacerun:yes'> </span>take the 2 restriction
 +
endonuclease cuts from 37c n run gel (1.5 gel 65v 45 min) <span class=SpellE>shd</span>
 +
be ~5500bp and ~3500bp<o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
 +
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
 +
EN-US'><o:p>&nbsp;</o:p></span></p>
 +
 +
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
 +
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
 +
EN-US'>11/9<o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
 +
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
 +
EN-US'>PCR clean for 3 in 1 cut<o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
 +
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
 +
EN-US'>Run gel for the dcas9 <span class=SpellE>gfp</span> <span class=SpellE>sgrna</span>
 +
no. 3 restriction endonuclease cut for dcas9 <span class=SpellE>gfp</span> <span
 +
class=SpellE>sgrna</span> (<span class=SpellE>XbaI</span>, <span class=SpellE>XhoI</span>),
 +
cut the 5500 band (65v 45 min) (big well) (all in)<o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
 +
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
 +
EN-US'>Gel purification for the above cut gel (gm buffer 650ul)<o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
 +
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
 +
EN-US'>Run gel for colony PCR yesterday (1.5 gel)<o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
 +
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
 +
EN-US'>Set ligation mixture (restriction endonuclease cuts <span class=GramE>of<span
 +
style='mso-spacerun:yes'>  </span>3</span> in 1 and <span class=SpellE>mcy</span>
 +
dcas9 <span class=SpellE>gfp</span>) 1:1<o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
 +
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
 +
EN-US'><o:p>&nbsp;</o:p></span></p>
 +
 +
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
 +
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
 +
EN-US'>12/9<o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
 +
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
 +
EN-US'>Heat inactivate ligation<o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
 +
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
 +
EN-US'>2. Transform ligation (total <span class=SpellE>dna</span> 60ng)<o:p></o:p></span></p>
 +
 +
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
 +
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
 +
EN-US'>3. Dilute overnight inoculation, 1 to 3<o:p></o:p></span></p>
  
−
<p>
+
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
 +
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
 +
EN-US'>4. Set restriction endonuclease cut of 3 in 1 and <span class=SpellE>sgrna</span>
 +
dcas9 <span class=SpellE>gfp</span><o:p></o:p></span></p>
  
−
    <strong> </strong>
+
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
−
</p>
+
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
−
<p>
+
EN-US'><o:p>&nbsp;</o:p></span></p>
  
−
    <strong> </strong>
+
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
−
</p>
+
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
−
<p>
+
EN-US'>13/9<o:p></o:p></span></p>
  
−
        <strong>4) Transformed Gib 4.1 Gib 4.2</strong>
+
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
−
</p>
+
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
−
<p>
+
EN-US'>Measure od (growth curve) for modelling<o:p></o:p></span></p>
  
−
    <strong>Overnight ligation sgRNA mcyI Petblue</strong>
+
<p class=MsoNormal style='tab-stops:5.0cm'><span lang=EN-US style='font-family:
−
</p>
+
Times;mso-bidi-font-family:"Times New Roman";mso-ansi-language:EN-US'><span
−
<p>
+
style='mso-tab-count:1'>                                              </span></span><span
 +
lang=EN-US style='font-family:"Times New Roman",serif;mso-fareast-font-family:
 +
"Times New Roman";mso-ansi-language:EN-US'><o:p></o:p></span></p>
  
−
    <strong>sgRNA mcyB petblue</strong>
+
<p class=MsoNormal style='tab-stops:5.0cm'><span lang=EN-US style='font-family:
−
</p>
+
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
−
<p>
+
EN-US'>16/9<o:p></o:p></span></p>
  
−
    <strong> Gib -0.9uL DNA </strong>
+
<p class=MsoNormal style='tab-stops:5.0cm'><span lang=EN-US style='font-family:
−
</p>
+
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
−
<p>
+
EN-US'>Restriction endonuclease cuts for final product (<span class=SpellE>XhoI</span>,
 +
<span class=SpellE>XbaI</span>)<o:p></o:p></span></p>
  
−
    <strong> sgRNA -2uL</strong>
+
<p class=MsoNormal style='tab-stops:5.0cm'><span lang=EN-US style='font-family:
−
</p>
+
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
−
<p>
+
EN-US'>Pick colony for growth curve<o:p></o:p></span></p>
  
−
    <strong> </strong>
+
<p class=MsoNormal style='tab-stops:5.0cm'><span lang=EN-US style='font-family:
−
</p>
+
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
−
<p>
+
EN-US'><o:p>&nbsp;</o:p></span></p>
  
−
        <strong>5) Checked plates for previous transformation: Gib 3 in 1 -</strong>
+
<p class=MsoNormal style='tab-stops:5.0cm'><span lang=EN-US style='font-family:
−
</p>
+
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
−
<p>
+
EN-US'>17/9<o:p></o:p></span></p>
  
−
    <strong>                                                                         Gib 2  - No colonies</strong>
+
<p class=MsoNormal style='tab-stops:5.0cm'><span lang=EN-US style='font-family:
−
</p>
+
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
−
<p>
+
EN-US'>Growth curve<o:p></o:p></span></p>
  
−
    <strong>                                                                         Gib 3        -</strong>
+
<p class=MsoNormal style='tab-stops:5.0cm'><span lang=EN-US style='font-family:
−
</p>
+
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
−
<p>
+
EN-US'>Run gel for the restriction endonuclease cuts, PCR clean, start ligation<o:p></o:p></span></p>
  
−
    <strong> From last yr -petblue stock -yes colonies</strong>
+
<p class=MsoNormal style='tab-stops:5.0cm'><span lang=EN-US style='font-family:
−
</p>
+
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
−
<p>
+
EN-US'>Transform ligation mixture<o:p></o:p></span></p>
  
−
    <strong> </strong>
+
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
−
</p>
+
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
−
<p>
+
EN-US'><o:p>&nbsp;</o:p></span></p>
  
−
        <strong>6) Picked 6 colonies from petblue</strong>
+
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
−
</p>
+
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
−
<p>
+
EN-US'>18/9<o:p></o:p></span></p>
  
−
    <strong>             Petblue 1-3 -3mL    (with</strong>
+
<p class=MsoNormal style='tab-stops:283.0pt'><span class=SpellE><span
−
</p>
+
lang=EN-US style='font-family:"Times New Roman",serif;mso-fareast-font-family:
−
<p>
+
"Times New Roman";mso-ansi-language:EN-US'>Transofrmation</span></span><span
 +
lang=EN-US style='font-family:"Times New Roman",serif;mso-fareast-font-family:
 +
"Times New Roman";mso-ansi-language:EN-US'> of final ligation products<o:p></o:p></span></p>
  
−
    <strong>             Petblue 4-5 -2mL  amp)</strong>
+
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
−
</p>
+
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
−
<p>
+
EN-US'>Od measurements<o:p></o:p></span></p>
  
−
    <strong>Master plate -No xgal Iptg</strong>
+
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
−
</p>
+
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
−
<p>
+
EN-US'><o:p>&nbsp;</o:p></span></p>
  
−
    <strong> </strong>
+
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
−
</p>
+
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
−
<p>
+
EN-US'>19/9<o:p></o:p></span></p>
  
−
        <strong>7) Run gel for pcr products</strong>
+
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
−
</p>
+
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
−
<p>
+
EN-US'>pick colony (fat clone)<o:p></o:p></span></p>
  
−
    <strong>Frag 2, frag 3 X Gib</strong>
+
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
−
</p>
+
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
−
<p>
+
EN-US'>Transformation final product<o:p></o:p></span></p>
  
−
    <strong> </strong>
+
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
−
</p>
+
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
−
<p>
+
EN-US'><o:p>&nbsp;</o:p></span></p>
  
−
    <strong>Gel purification</strong>
+
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
−
</p>
+
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
−
<p>
+
EN-US'>20/9<o:p></o:p></span></p>
  
−
    <strong>DNA concentration on 13/8</strong>
+
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
−
</p>
+
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
−
<p>
+
EN-US'>Pick clone<o:p></o:p></span></p>
  
−
    <strong> </strong>
+
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
−
</p>
+
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
−
<p>
+
EN-US'>PCR check band (Correct band at 12kbp)<o:p></o:p></span></p>
  
−
    <strong> </strong>
+
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
−
</p>
+
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
−
<p>
+
EN-US'>Plasmid purification (Takara)<o:p></o:p></span></p>
  
−
    <strong> </strong>
+
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
−
</p>
+
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
−
<p>
+
EN-US'><o:p>&nbsp;</o:p></span></p>
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong>15 August</strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
  
−
        <strong>1) RE cut for frag1 frag 2 and frag 3</strong>
+
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
−
</p>
+
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
−
<p>
+
EN-US'><o:p>&nbsp;</o:p></span></p>
  
−
    <strong>X Gib ~6 hr</strong>
+
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
−
</p>
+
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
−
<p>
+
EN-US'><o:p>&nbsp;</o:p></span></p>
−
<strong>        </strong>
+
−
</p>
+
−
<p>
+
−
<strong>        Nanodrop before RE cut</strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
  
−
        <strong>2) Set up Gibson reaction</strong>
+
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
−
</p>
+
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
−
<p>
+
EN-US'>23/9<o:p></o:p></span></p>
  
−
            <strong>a. Frag 3 50nL 16uL              -       </strong>
+
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
−
</p>
+
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
−
<p>
+
EN-US'>Pick colony of final product<o:p></o:p></span></p>
  
−
            <strong>b. Frag 2 38.73ng 2.1 uL        -</strong>
+
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
−
</p>
+
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
−
<p>
+
EN-US'><o:p>&nbsp;</o:p></span></p>
  
−
            <strong>c. Frag 1 29.26ng 0.4uL        -       20uL/xh</strong>
+
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
−
</p>
+
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";color:black;
−
<p>
+
mso-themecolor:text1;mso-ansi-language:EN-US'>24/9<o:p></o:p></span></p>
  
−
            <strong>d. Nebuilder          10uL          -</strong>
+
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
−
</p>
+
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
−
<p>
+
EN-US'>Plasmid purification (Takara)<o:p></o:p></span></p>
  
−
            <strong>e. H2O              5.9uL        -</strong>
+
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
−
</p>
+
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
−
<p>
+
EN-US'><o:p>&nbsp;</o:p></span></p>
  
−
        <strong> </strong>
+
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
−
</p>
+
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
−
<p>
+
EN-US'>25/9 <o:p></o:p></span></p>
  
−
        <strong>50C for 2hr</strong>
+
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
−
</p>
+
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
−
<p>
+
EN-US'>Nanodrop final product<o:p></o:p></span></p>
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
  
−
        <strong>3) Plasmid Purification of Petblue</strong>
+
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
−
</p>
+
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";color:black;
−
<p>
+
mso-themecolor:text1;mso-ansi-language:EN-US'><o:p>&nbsp;</o:p></span></p>
−
<strong>                    Elude using 30uL</strong>
+
−
</p>
+
−
<p>
+
−
<strong>        Nanodrop</strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
  
−
        <strong>4) Gib 4.1 -Petblue mcyB plasmid have 1 colony each</strong>
+
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
−
</p>
+
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";color:black;
−
<p>
+
mso-themecolor:text1;mso-ansi-language:EN-US'>26/9<o:p></o:p></span></p>
−
<strong>                    -Picked & inoculated</strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
  
−
        <strong>5) PCR purified RE cuts from (nolning?)</strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Restriction
−
<p>
+
endonuclease cut for final product (<span class=SpellE>PstI</span>)<o:p></o:p></span></p>
  
−
    <strong>-ligated H3 & 3 in 1</strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'><o:p>&nbsp;</o:p></span></p>
−
<p>
+
  
−
    <strong>        1+3  1:1</strong>
+
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
−
</p>
+
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
−
<p>
+
EN-US'>27/9<o:p></o:p></span></p>
  
−
    <strong>       1+2+3 1:1:1</strong>
+
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
−
</p>
+
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
−
<p>
+
EN-US'>Run gel for the restriction endonuclease cuts and final product 24 and
 +
15. <o:p></o:p></span></p>
  
−
    <strong> </strong>
+
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
−
</p>
+
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
−
<p>
+
EN-US'>All in for restriction endonuclease cuts, <o:p></o:p></span></p>
  
−
        <strong>6) Sequencing</strong>
+
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
−
</p>
+
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
−
<p>
+
EN-US'>5ul for final product 24 and 15<o:p></o:p></span></p>
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong>16 August</strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
  
−
        <strong>1) Plasmid purification of</strong>
+
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
−
</p>
+
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
−
<p>
+
EN-US'>65v 50min<o:p></o:p></span></p>
  
−
    <strong>Gib4.1 & pET blue mcyB plasmid from 1518</strong>
+
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
−
</p>
+
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
−
<p>
+
EN-US'>Nanodrop final product<o:p></o:p></span></p>
  
−
    <strong>Gib4.1 does not seem to grow</strong>
+
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
−
</p>
+
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
−
<p>
+
EN-US'><o:p>&nbsp;</o:p></span></p>
  
−
    <strong>During inoculation</strong>
+
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
−
</p>
+
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
−
<p>
+
EN-US'>30/9<o:p></o:p></span></p>
  
−
    <strong>Therefore, low yield.</strong>
+
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
−
</p>
+
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
−
<p>
+
EN-US'>Transformation<o:p></o:p></span></p>
  
−
    <strong> </strong>
+
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
−
</p>
+
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
−
<p>
+
EN-US'><span style='mso-spacerun:yes'> </span>final product plasmid 15 17 24
 +
29(BL21)<o:p></o:p></span></p>
  
−
        <strong>2) Run gel for frag 1+3 & 3in1 (ligated)</strong>
+
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
−
</p>
+
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
−
<p>
+
EN-US'><o:p>&nbsp;</o:p></span></p>
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
  
−
        <strong>3) Intended to amp 1+3 using 3F + 1R and 3R + 1F</strong>
+
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
−
</p>
+
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
−
<p>
+
EN-US'><o:p>&nbsp;</o:p></span></p>
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
  
−
        <strong>4) Intended to check Gib4 using 1+2, 2+3, 1+3 primers.</strong>
+
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
−
</p>
+
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
−
<p>
+
EN-US'>3/10<o:p></o:p></span></p>
  
−
    <strong> </strong>
+
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
−
</p>
+
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
−
<p>
+
EN-US'>Observe green fluorescence by UV exposure<o:p></o:p></span></p>
  
−
    <strong> </strong>
+
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
−
</p>
+
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
−
<p>
+
EN-US'>Inoculate<o:p></o:p></span></p>
−
<strong>(3) and (4) failed possibily due to pcr errors</strong>
+
−
</p>
+
−
<p>
+
−
<strong>-Use 3 mins extension        65C other times and maximum</strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
  
−
        <strong>5) Intended to check sgRNA mcyB petblue using colony PCR</strong>
+
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
−
</p>
+
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
−
<p>
+
EN-US'>Observe green fluorescence again by UV exposure<o:p></o:p></span></p>
  
−
    <strong>(failed due to 0.7% gel, not 1.5% gel pic in chelsy’s phone</strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>All negative
−
<p>
+
</span><span lang=EN-US style='font-family:Times;mso-ansi-language:EN-US'><o:p></o:p></span></p>
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong>19 August</strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
  
−
        <strong>1) Colony PCR for McyB sgRNA petblue</strong>
+
<p class=MsoNormal style='margin-left:18.0pt'><span lang=EN-US
−
</p>
+
style='font-family:Times;mso-ansi-language:EN-US'><o:p>&nbsp;</o:p></span></p>
−
<p>
+
  
−
        <strong>(only 1 white colony)</strong>
+
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
−
</p>
+
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";color:black;
−
<p>
+
mso-themecolor:text1;mso-ansi-language:EN-US'>4/10<o:p></o:p></span></p>
  
−
        <strong>Results positive: band size ~300bp</strong>
+
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
−
</p>
+
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";color:black;
−
<p>
+
mso-themecolor:text1;mso-ansi-language:EN-US'>Plasmid purification of final
 +
product<o:p></o:p></span></p>
  
−
        <strong> </strong>
+
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
−
</p>
+
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";color:red;
−
<p>
+
mso-ansi-language:EN-US'><o:p>&nbsp;</o:p></span></p>
  
−
        <strong>2) Inoculated 2 3mL tubes of Gib4</strong>
+
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
−
</p>
+
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";color:black;
−
<p>
+
mso-themecolor:text1;mso-ansi-language:EN-US'>7/10<o:p></o:p></span></p>
  
−
        <strong>-Gib4.1 & 4.2</strong>
+
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
−
</p>
+
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";color:black;
−
<p>
+
mso-themecolor:text1;mso-ansi-language:EN-US'>Transformation of final products
 +
into DH5</span><span lang=EN-US style='font-family:Symbol;mso-fareast-font-family:
 +
"Times New Roman";mso-bidi-font-family:"Times New Roman";color:black;
 +
mso-themecolor:text1;mso-ansi-language:EN-US'>a<o:p></o:p></span></p>
  
−
        <strong>From master plate</strong>
+
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
−
</p>
+
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
−
<p>
+
EN-US'><o:p>&nbsp;</o:p></span></p>
  
−
        <strong> </strong>
+
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
−
</p>
+
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
−
<p>
+
EN-US'>8/10<o:p></o:p></span></p>
  
−
        <strong>3) Set up overnight PCR to amp mcyI, frag1, frag3 xgib,</strong>
+
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
−
</p>
+
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
−
<p>
+
EN-US'>Prepared 0.1mm <span class=SpellE>nacl</span>, 0.1mm <span class=SpellE>hepes</span>,
 +
0.1mm vitb12, all not sterilized. Picked 10 colonies (final product<span
 +
class=GramE>) ,</span> inoculated in 20ml LB <o:p></o:p></span></p>
  
−
    <strong> </strong>
+
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
−
</p>
+
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
−
<p>
+
EN-US'><o:p>&nbsp;</o:p></span></p>
  
−
        <strong>4) RE cut mcyI (247.8ng), petblue (1000ng), frag1 (741ng)- frag3 (140ng) overnight [McyI only added 3uL]</strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";color:black;mso-themecolor:text1;
−
<p>
+
mso-ansi-language:EN-US'>9/10</span><span lang=EN-US style='font-family:Times;
 +
mso-bidi-font-family:"Times New Roman";color:black;mso-themecolor:text1;
 +
mso-ansi-language:EN-US'><o:p></o:p></span></p>
  
−
    <strong> </strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";color:black;mso-themecolor:text1;
−
<p>
+
mso-ansi-language:EN-US'>Filtered Vitamin B12<o:p></o:p></span></p>
  
−
        <strong>5) Checked Neb 3 in 1 using different primers</strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";color:black;mso-themecolor:text1;
−
<p>
+
mso-ansi-language:EN-US'>Autoclave bold3n<o:p></o:p></span></p>
  
−
    <strong>Checked frag 1+3</strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";color:black;mso-themecolor:text1;
−
<p>
+
mso-ansi-language:EN-US'>Add to agar</span><span lang=EN-US style='font-size:
−
<strong> </strong>
+
11.0pt;font-family:"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";
−
</p>
+
color:black;mso-themecolor:text1;mso-ansi-language:EN-US'><o:p></o:p></span></p>
−
<p>
+
−
<strong> 20 August</strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
  
−
        <strong>1) Run gel for mcyI, frag1-3 xGib</strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";color:black;mso-themecolor:text1;
−
<p>
+
mso-ansi-language:EN-US'><o:p>&nbsp;</o:p></span></p>
  
−
    <strong>-gel purification Takara</strong>
+
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
−
</p>
+
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
−
<p>
+
EN-US'>10/10<o:p></o:p></span></p>
  
−
    <strong> </strong>
+
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
−
</p>
+
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
−
<p>
+
EN-US'>Plasmid purification with ethanol <span class=SpellE>percipitation</span><o:p></o:p></span></p>
  
−
        <strong>2) PCR clean mcyI, petblue 2 from overnight RE</strong>
+
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
−
</p>
+
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
−
<p>
+
EN-US'><o:p>&nbsp;</o:p></span></p>
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
  
−
        <strong>3) Ligated mcyI petblue, frag1+3</strong>
+
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
−
</p>
+
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
−
<p>
+
EN-US'><o:p>&nbsp;</o:p></span></p>
  
−
    <strong>-vector 100ng  -vector 100ng</strong>
+
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
−
</p>
+
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
−
<p>
+
EN-US'>11/10<o:p></o:p></span></p>
−
<strong>        1:3                      1:1</strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
  
−
        <strong>4) ?</strong>
+
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
−
</p>
+
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
−
<p>
+
EN-US'>Measure Optical density (growth curve)<o:p></o:p></span></p>
  
−
    <strong> </strong>
+
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
−
</p>
+
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
−
<p>
+
EN-US'>Transformation of Microcystis<o:p></o:p></span></p>
  
−
        <strong>5) Plasmid pur Gib6.4</strong>
+
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
−
</p>
+
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
−
<p>
+
EN-US'>Electroporation<o:p></o:p></span></p>
  
−
    <strong>-yield extremely low</strong>
+
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
−
</p>
+
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
−
<p>
+
EN-US'>Ligation by Peter<o:p></o:p></span></p>
  
−
    <strong> </strong>
+
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
−
</p>
+
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
−
<p>
+
EN-US'>DNA purification <o:p></o:p></span></p>
  
−
        <strong>6) Picked colonies: 2 Nebuilder 3in1.           -</strong>
+
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
−
</p>
+
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
−
<p>
+
EN-US'><o:p>&nbsp;</o:p></span></p>
−
<strong>                                  2 Gib4                          -       Master Plate</strong>
+
−
</p>
+
−
<p>
+
−
<strong>                                  2 sgRNA mcyB petblue  -</strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong>21 August</strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
  
−
        <strong>1) Plasmid pur for 6 tubes -NEbuilder x2, gib4 x2, sgRNA mcyB petblue</strong>
+
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
−
</p>
+
Times;mso-bidi-font-family:"Times New Roman";mso-ansi-language:EN-US'>14/10</span><span
−
<p>
+
lang=EN-US style='font-family:"Times New Roman",serif;mso-fareast-font-family:
−
<strong> </strong>
+
"Times New Roman";mso-ansi-language:EN-US'><o:p></o:p></span></p>
−
</p>
+
−
<p>
+
  
−
        <strong>2) Collected overnight ligation</strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Transformation
−
<p>
+
of previous ligation<o:p></o:p></span></p>
  
−
    <strong>-Amplified frag 1+3 wing</strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'><o:p>&nbsp;</o:p></span></p>
−
<p>
+
−
<strong>Primer frag1 F & primer frag3 R (vice versa also)</strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
  
−
        <strong>3) Transformed sgRNA mcyI petblue and dCas9 gfp (L3)</strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>15/10<o:p></o:p></span></p>
−
<p>
+
  
−
    <strong> </strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Pick clones,
−
<p>
+
inoculate i12<o:p></o:p></span></p>
  
−
        <strong>4) Received sanger sequence results</strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Standard
−
<p>
+
solution<o:p></o:p></span></p>
  
−
    <strong>-99.4% identical</strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'><o:p>&nbsp;</o:p></span></p>
−
<p>
+
  
−
    <strong>       Mutation- X</strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>16/10<o:p></o:p></span></p>
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
  
−
        <strong>5) Picked 25 colonies from previous L3 plate</strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Pour plate
−
<p>
+
with kanamycin resistance and chloramphenicol<span style='mso-spacerun:yes'>  
 +
</span><o:p></o:p></span></p>
  
−
    <strong>-colony PCR          25-49  -one Taq</strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Takara
−
<p>
+
plasmid purification of insert 1 and insert 2<o:p></o:p></span></p>
  
−
    <strong>                               50    - sapphire</strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'><o:p>&nbsp;</o:p></span></p>
−
<p>
+
  
−
    <strong> </strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>17/10<o:p></o:p></span></p>
−
<p>
+
  
−
        <strong>6) Single cut & double cut 3 in 1 plasmid</strong>
+
<p class=MsoNormal style='line-height:107%'><span lang=EN-US style='font-family:
−
</p>
+
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
−
<p>
+
EN-US'>Peter ethanol precipitation of final product<o:p></o:p></span></p>
  
−
    <strong>-All undesired results</strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Electroporation
−
<p>
+
of Microcystis Aeruginosa UTEX 2388<o:p></o:p></span></p>
  
−
    <strong> </strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Ligation
−
<p>
+
mixture (cut with <span class=SpellE>SpelI</span>, <span class=SpellE>xbaI</span>)<o:p></o:p></span></p>
−
<strong> 22aug</strong>
+
−
</p>
+
−
<p>
+
−
<strong> Gibson assembly ,incubate 2h</strong>
+
−
</p>
+
−
<p>
+
−
<strong> Run gel for colony pcr and frag 1+3 and the overnight cut of 3 in 1</strong>
+
−
</p>
+
−
<p>
+
−
<strong>For colonies with correct band after colony pcr, pick colony from master plate, inoculate</strong>
+
−
</p>
+
−
<p>
+
−
<strong>Frag 1+3, frag 2 re cut,</strong>
+
−
</p>
+
−
<p>
+
−
<strong>Send mcyB to sequencing</strong>
+
−
</p>
+
−
<p>
+
−
<strong>pick colony from plates  if have</strong>
+
−
</p>
+
−
<p>
+
−
<strong>Transform gibson products</strong>
+
−
</p>
+
−
<p>
+
−
<strong>Ligate frag 2 to 1+3</strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong>23 aug</strong>
+
−
</p>
+
−
<p>
+
−
<strong>Run gel for Pcr dcas9 gfp,</strong>
+
−
</p>
+
−
<p>
+
−
<strong>Run gel for Pcr 1+3 sgrna mcyI pet blue</strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong>26 aug</strong>
+
−
</p>
+
−
<p>
+
−
<strong>Run gel for previous 40 colony pcr</strong>
+
−
</p>
+
−
<p>
+
−
<strong>Inoculate L3 with correct band size</strong>
+
−
</p>
+
−
<p>
+
−
<strong>Preparation for electroporation</strong>
+
−
</p>
+
−
<p>
+
−
<strong>Transform ligation product and  3 in 1 for characterization</strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong>27 aug</strong>
+
−
</p>
+
−
<p>
+
−
<strong>Pick clone for previous transformation</strong>
+
−
</p>
+
−
<p>
+
−
<strong>3 in 1 plasmid pur using traditional method</strong>
+
−
</p>
+
−
<p>
+
−
<strong>Re cut gib 5</strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong>28 aug</strong>
+
−
</p>
+
−
<p>
+
−
<strong>PCR screening mcyI, B</strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong>29 aug</strong>
+
−
</p>
+
−
<p>
+
−
<strong>Plasmid pur. Using tradition n takara</strong>
+
−
</p>
+
−
<p>
+
−
<strong>Inoculate gib 5, prepare for measurement</strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong>30 aug</strong>
+
−
</p>
+
−
<p>
+
−
<strong>I think no lab</strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong>31 aug</strong>
+
−
</p>
+
−
<p>
+
−
<strong>Pick sgRNA clone?</strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong>1/9</strong>
+
−
</p>
+
−
<p>
+
−
<strong>I think no lab</strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong>2/9</strong>
+
−
</p>
+
−
<p>
+
−
<strong>I think no lab</strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong>3/9</strong>
+
−
</p>
+
−
<p>
+
−
<strong>Re cut dcas9 gfp</strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong>4/9</strong>
+
−
</p>
+
−
<p>
+
−
<strong>Run gel for re cut dcas9 gfp</strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong>5/9</strong>
+
−
</p>
+
−
<p>
+
−
<strong>Transformation inoculate pet blue, puc19, 3 in 1, psb1c3 transformants</strong>
+
−
</p>
+
−
<p>
+
−
<strong>Plasmid purification 3 in 1 using Takara plasmid purification kit</strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong>6/9</strong>
+
−
</p>
+
−
<p>
+
−
<strong>Plasmid purification 3 in 1 using takara plasmid purification kit</strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong>9/9</strong>
+
−
</p>
+
−
<p>
+
−
<strong>2 re cuts</strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong>10/9</strong>
+
−
</p>
+
−
<p>
+
−
<strong>run gel for the colony pcr products (1.5 gel, all in) 65V 45 min 700 or no band</strong>
+
−
</p>
+
−
<p>
+
−
<strong>Pick colony and colony pcr</strong>
+
−
</p>
+
−
<p>
+
−
<strong>pour agar plate</strong>
+
−
</p>
+
−
<p>
+
−
<strong> Take the 2 re cuts from 37C n run gel (1.5 gel 65V 45 min) shd be ~5500 and ~3500</strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong>11/9</strong>
+
−
</p>
+
−
<p>
+
−
<strong>PCR clean for 3 in 1 cut</strong>
+
−
</p>
+
−
<p>
+
−
<strong>Run gel for the dcas9 gfp sgrna no. 3 re cut for dcas9 gfp sgrna, cut the 5500 band (65V 45 min) (big well) (all in)</strong>
+
−
</p>
+
−
<p>
+
−
<strong>Gel pur for the above cut gel (GM buffer 650ul)</strong>
+
−
</p>
+
−
<p>
+
−
<strong>Run gel for colony pcr yesterday (1.5 gel)</strong>
+
−
</p>
+
−
<p>
+
−
<strong>Set ligation mixture (re cuts of  3 in 1 and Mcy dcas9 gfp) 1:1</strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong>12/9</strong>
+
−
</p>
+
−
<p>
+
−
<strong>Heat inactivate ligation</strong>
+
−
</p>
+
−
<p>
+
−
<strong>2. Transform ligation (total dna 60ng)</strong>
+
−
</p>
+
−
<p>
+
−
<strong>3. Dilute overnight inoculation, 1 to 3</strong>
+
−
</p>
+
−
<p>
+
−
<strong>4. Set RE of 3 in 1 and sgrna dcas9 gfp</strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong>13/9</strong>
+
−
</p>
+
−
<p>
+
−
<strong>Measure OD (growth curve) for modelling</strong>
+
−
</p>
+
−
<p>
+
−
<strong>                                          </strong>
+
−
</p>
+
−
<p>
+
−
<strong>16/9</strong>
+
−
</p>
+
−
<p>
+
−
<strong>Re cuts for final product</strong>
+
−
</p>
+
−
<p>
+
−
<strong>pick colony for growth curve</strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong>17/9</strong>
+
−
</p>
+
−
<p>
+
−
<strong>growth curve</strong>
+
−
</p>
+
−
<p>
+
−
<strong>Run gel for the RE cuts, pcr clean, start ligation</strong>
+
−
</p>
+
−
<p>
+
−
<strong>transform ligation mixture</strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong>18/9</strong>
+
−
</p>
+
−
<p>
+
−
<strong>Transofrmation of final ligation products</strong>
+
−
</p>
+
−
<p>
+
−
<strong>2. OD measurements</strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong>19/9</strong>
+
−
</p>
+
−
<p>
+
−
<strong> Pick colony (fat Clone)</strong>
+
−
</p>
+
−
<p>
+
−
<strong>Transformation final product</strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong>20/9</strong>
+
−
</p>
+
−
<p>
+
−
<strong>Pick clone</strong>
+
−
</p>
+
−
<p>
+
−
<strong>Pcr check band</strong>
+
−
</p>
+
−
<p>
+
  
−
        <strong>·  Correct </strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Run gel for
−
<p>
+
plasmid purification product of I2<o:p></o:p></span></p>
−
<strong>Plasmid pur by wch failed cuz me filp his gel..</strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong>23/9</strong>
+
−
</p>
+
−
<p>
+
−
<strong>pick colony of final product</strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong>24/9</strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong>25/9</strong>
+
−
</p>
+
−
<p>
+
−
<strong>Nanodrop final product</strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong>26/9</strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong>27/9</strong>
+
−
</p>
+
−
<p>
+
−
<strong>run gel for the RE cuts and final product 24 and 15.</strong>
+
−
</p>
+
−
<p>
+
−
<strong>All in for re cuts,</strong>
+
−
</p>
+
−
<p>
+
−
<strong>5ul for final product 24 and 15</strong>
+
−
</p>
+
−
<p>
+
−
<strong>65V 50min</strong>
+
−
</p>
+
−
<p>
+
−
<strong>Nanodrop final product</strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong>30/9</strong>
+
−
</p>
+
−
<p>
+
−
<strong>transformation</strong>
+
−
</p>
+
−
<p>
+
−
<strong> final product plasmid 15 17 24 29(BL21)</strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong>1/10</strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong>2/10</strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong>3/10</strong>
+
−
</p>
+
−
<p>
+
−
<strong>observe green fluorescence by uv exposure</strong>
+
−
</p>
+
−
<p>
+
−
<strong>inoculate</strong>
+
−
</p>
+
−
<p>
+
−
<strong>observe green fluorescence again by uv exposure</strong>
+
−
</p>
+
−
<p>
+
  
−
        <strong>·  All negative</strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Nanodrop I2<o:p></o:p></span></p>
−
<p>
+
−
<strong>4/10</strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong>7/10</strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong>8/10</strong>
+
−
</p>
+
−
<p>
+
−
<strong>Prepared 0.1mM NaCl, 0.1mM HEPES, 0.1mM Vitb12, all not sterilized. Picked 10 colonies, inoculated in 20mL Lb</strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong>10/10</strong>
+
−
</p>
+
−
<p>
+
−
<strong>Plasmid purification with ethanol</strong>
+
−
</p>
+
−
<p>
+
−
<strong>Filter b12</strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong>11/10</strong>
+
−
</p>
+
−
<p>
+
−
<strong>OD (growth curve)</strong>
+
−
</p>
+
−
<p>
+
−
<strong>Transformation of microcystis</strong>
+
−
</p>
+
−
<p>
+
−
<strong>Electroporation</strong>
+
−
</p>
+
−
<p>
+
−
<strong>Ligation by wch</strong>
+
−
</p>
+
−
<p>
+
−
<strong>Dna purification</strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong>16/10</strong>
+
−
</p>
+
−
<p>
+
−
<strong>Pour plate with kanamycin resistance and chl </strong>
+
−
</p>
+
−
<p>
+
−
<strong>Plasmid Purification of Insert 1 and Insert 2</strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong>18/10</strong>
+
−
</p>
+
−
<p>
+
−
<strong>Pick Insert 1 and 2 ,BL21 and NEB5a clones</strong>
+
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
−
<strong>Missing</strong>
+
−
</p>
+
−
<p>
+
  
−
        <strong>1.    20-21/7</strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Anson adjust
−
<p>
+
pH for BG11<o:p></o:p></span></p>
  
−
        <strong>2.    23/7-8/8</strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Error: labelling
−
<p>
+
error for plasmid purification<o:p></o:p></span></p>
  
−
        <strong>3.    24/9 (no gel pic n whatsapp)</strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'><o:p>&nbsp;</o:p></span></p>
−
<p>
+
  
−
        <strong>4.    26/9 (no gel pic n whatsapp)</strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>18/10<o:p></o:p></span></p>
−
<p>
+
  
−
        <strong>5.    1/10 (no gel pic n whatsapp)</strong>
+
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Pick insert
−
<p>
+
1 and <span class=GramE>2 ,BL</span>21 and NEB5</span><span lang=EN-US
 +
style='font-family:Symbol;mso-fareast-font-family:Symbol;mso-bidi-font-family:
 +
Symbol;mso-ansi-language:EN-US'>a</span><span lang=EN-US style='font-family:
 +
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
 +
EN-US'> clones <o:p></o:p></span></p>
  
−
        <strong>6.    2/10(no gel pic n whatsapp)</strong>
+
<p class=MsoListParagraph><span lang=EN-US style='font-family:"Times New Roman",serif;
−
</p>
+
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'><o:p>&nbsp;</o:p></span></p>
−
<p>
+
  
−
        <strong>7.    4/10(no gel pic n whatsapp)</strong>
+
</div>
−
</p>
+
−
<p>
+
  
−
        <strong>8.    7/10(no gel pic n whatsapp)</strong>
+
</body>
−
</p>
+
−
<p>
+
−
<strong> </strong>
+
−
</p>
+
−
+
  
 
</html>
 
</html>

Revision as of 02:10, 22 October 2019

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JUDGING FORM ⇗

17 July

Transformation of dcas9 plasmid 2019 kit plate 5 well 22N

to E. coli NEB5a using kanamycin resistance plate

 

19 July

Picked 6 colonies

grow overnight in LB

Plasmid purification (Takara)

colony 1 & 2 did not grow

run agarose gel for 100v 30 mins

eluted DNA using 30ul elution buffer

nanodrop the eluted DNA

 

22 July

Plasmid prep

Run gel for dCas9

Resuspend DNA

 

23 July

Assembling Fragment 1+2+3

restriction endonuclease cut + ligation

Using NheI and BssSI-v2

Dcas9 preparation clean

Run gel dCas9

Transformed produce to E.coli neb5a

Cloned GFP to dcas9

restriction endonuclease cut + ligation

Using EcorI NsiI

Transformed product to E.coli neb5a

Transformed puc19 from stock (for cloning sgrna later)

 

1 Aug

Cyanobacteria arrived, cultured in 15x4 ml medium

GFP-dcas9 plasmid ligation (all failed)

 

2 Aug

Colony PCR for L2, 3, 4

PCR Fragment 1, 2, 3

Plasmid purification

 

6 Aug

colony PCR

standard q5 PCR preperation for gibson

Gibson assembly

GFP+ dCas9 plasmid again as L3 Failed

 

 

 

 

7 Aug

gel purification after 75v 45min 0.7 gel

gibson assembly

run gel for overnight sgrna PCR product

restriction endonuclease cut (PstI, HindIII)(2hrs incubation)+ligation for sgRNA and PETblue

transformed 3in1, PETblue sgRNA and L3 into NEB5a

 

8 Aug

Colony PCR for l3

Run gel for 3in1 (1 smear),

Pick clone

 

9 August

Checked plates for sgRNA PETblue McyI(no colony)

sgRNA pet blue McyI(no colony)

sgRNA pet blue Mcyb (no colony)

3 in 1(no colony)

Gib 2.1(no colony)

Gib 2.2(no colony)

Conducted PCR purification of overnight (14h) restriction endonuclease cut of dCas9 GFP using EcoRI, NsiI

50 μL rxn mixture

Conducted ligation

1:3 & 1:5

Vector 250ng

20 μL rxn mixture

Placed in room temperature for 10 min

heat inactive at 65°c for 10 min

on ice 90min

then add ligase again

ligate over weekend.

Amplified Fragment1 + 2 + 3

sgrna McyI, sgRNA McyB, GFP

Fragment 1 2 3 GFP, 65c

sgRNA 55c

All 50 μL rxn

Weight powder, for soc (800ml no glucose) (no yellow)

LB agar (have yellow) 1800ul

Glucose 9g

Run gel for all 6 Fragments (all in)

75V 45 mins

Ladder use 1kb & 50bp

Loading dye 6x

Gel purification -elute using 30ul elution buffer.

 

 

 

 

 

 

12 August

Colony PCR for those without bands reaction

picked colonies from master plate

Performed gibson(gib3) & nebuilder

Scaled down to 10ml rxn

PCR for xgib 1 (Fragment1) gib1 (Fragment1)

xgib 2 (Fragment2) gib2 (Fragment2)

xgib 3 (Fragment3) gib3 (Fragment3)

 

Colony PCR no.24 correct band

(dCas9 GFP) L3

band size 700-800 bp

inoculated 24 Hours (picked from master plate)

 

Transformed gibson 1 gibson 2 gibson 3.2 nebuilder (1)

+ve control petblue nebuilder (2)

dcas9 gfp 1:3

dcas9 gfp 1:5

gibson 3.1

 

13 August

Run gel for previous PCR

Checked plates from 12/8

Colony: PETblue +ve

dCas9 GFP 1:3

dCas9 GFP 1:5

Plasmid purification L3

dCas9-GFP 24

 

Restriction endonuclease for Fragment1-3 ( for gibson assembly)

McyI, McyB

 

14 August

Run gel for xgib Fragment 1

no bands at all

Possibly due to pipetting errors.

Started 10ul reaction using gibson concentration according to following gel. (pic gib4)

Reaction: Fragment3: 50ng

Fragment2: 38.73ng

Fragment1: 29.26ng

nebuilder: 5ul

h2o

 

1)    Set up PCR reaction for xgib Fragment2

Xgib Fragment3

 

98°c

98°c

66°c

72°c

72°c

4°c

30sec

5sec

15sec

1.5min

2min

¥

 

 

 

2)    Transformed gib 4.1 gib 4.2

Overnight ligation sgrna McyI petblue

Sgrna Mcyb PETblue

gib -0.9ul dna

sgrna -2ul

 

3)    Checked plates for previous transformation: gib 3 in 1 -

gib2 - no colonierom last year -PETblue stock - there are colonies

 

4)    Picked 6 colonies from PETblue

PETblue 1-3 -3ml (with amp)

PETblue 4-5 -2ml

Master plate -no xgal iptg

 

5)    Run gel for PCR products

Fragment 2, Fragment 3 xgib

Gel purification

DNA concentration on 13/8

 

15 August

 

Restriction endonuclease cut for Fragment1 Fragment 2 and Fragment 3

Xgib ~6 hr (MluI, BssaI and NheI)

nanodrop before restriction endonuclease cut

Set up gibson reaction

Fragment 3 50ng 16ul

Fragment 2 38.73ng 2.1 ul

Fragment 1 29.26ng 0.4ul

Nebuilder 10ul

H2o 5.9ul

50°c for 2hr (optimized protocol)

 

Plasmid purification of PETblue

eluted using 30ul elution buffer

nanodrop

 

Gib 4.1 -PETblue McyB plasmid have 1 colony each

picked & inoculated

 

 

PCR purified restriction endonuclease cuts from (Fragment 1, 2, 3)

ligated h3 & 3 in 1

1+3 1:1

1+2+3 1:1:1

Sequencing

 

 

16 August

 

Plasmid purification of

Gib4.1 & pet blue McyB plasmid from 1518

Gib4.1 does not seem to grow during inoculation therefore, low yield.

Run gel for Fragment 1+3 & 3in1 (ligated)

Intended to amp 1+3 using 3f + 1r and 3r + 1f

Intended to check gib4 using 1+2, 2+3, 1+3 primers.

3 and 4 failed possibly due to PCR errors

use 3 mins extension 65°c other times and maximum

Intended to check sgRNA Mcyb PETblue using colony PCR

(failed due to 0.7% gel)

 

19 August

Colony PCR for Mcyb sgRNA PETblue

(only 1 white colony)

Results positive: band size ~300bp

Inoculated 2 3ml tubes of gib4

gib4.1 & 4.2

From master plate

Set up overnight PCR to amp mcyI, Fragment1, Fragment3 xgib,

Restriction endonuclease (PstI, HindIII, MluI, BssaI, NheI) cut McyI (247.8ng), PETblue (1000ng), Fragment1 (741ng)- Fragment3 (140ng) overnight [McyI only added 3ul]

Checked neb 3 in 1 using different primers

Checked Fragment 1+3

 

20 August

Run gel for mcyI, Fragment1-3 xgib

gel purification Takara

PCR clean McyI, PETblue 2 from overnight restriction endonuclease cut

Ligated McyI PETblue, Fragment1+3

vector 100ng -vector 100ng

1:3 1:1

Plasmid purification gib6.4

yield extremely low

Picked colonies: 2 from nebuilder 3in1.

2 from gib4 -master plate

2 from sgrRNA McyB PETblue-

 

21 August

Plasmid purifucation for 6 tubes - nebuilder x2, gib4 x2, Sgrna Mcyb PETblue

Collected overnight ligation

amplified Fragment 1+3

Primer Fragment1 f & primer Fragment3 (vice versa also)

Transformed sgRNA McyI PETblue and dCas9 GFP (L3)

Received sanger sequence results

99.4% identical

Mutation

\ wrong

 

Picked 25 colonies from previous l3 plate

colony PCR 25-49 -One Taq

50 - Sapphire

Single cut & double cut 3 in 1 plasmid

all undesired results

 

22 Aug

Gibson assembly, incubate 2h

run gel for colony PCR and Fragment 1+3 and the overnight cut of 3 in 1

For colonies with correct band after colony PCR, pick colony from master plate, inoculate

Fragment 1+3, Fragment 2 re cut,

Send McyB to sequencing

Transform gibson products

Ligate Fragment 2 to 1+3

 

23 Aug

Run gel for PCR dCas9 GFP,

Run gel for PCR 1+3 sgRNA MCYI PETblue

 

26 Aug

Run gel for previous 40 colony PCR

Inoculate l3 with correct band size

Preparation for electroporation

Transform ligation product and 3 in 1 for characterization

 

27 Aug

Pick clone for previous transformation

3 in 1 plasmid purification using ethanol perpetration

Restriction endonuclease cut gib 5 (MluI)

 

28 Aug

PCR screening McyI, McyB

 

29 Aug

Plasmid purification. Using Takara

Inoculate gib 5, prepare for measurement

 

3/9

Restriction endonuclease cut dCas9 GFP (EcoRI, PstI)

 

4/9

Run gel for restriction endonuclease cut dCas9 GFP

 

5/9

Transformation inoculate PETblue, Puc19, 3 in 1, PsB1c3 transformants

Plasmid purification 3 in 1 using Takara plasmid purification kit

 

6/9

Plasmid purification 3 in 1 using Takara plasmid purification kit

 

9/9

2 restriction endonuclease cuts (dCas9-GFP-sgRNA)

 

10/9

Run gel for the colony PCR products (1.5 gel, all in) 65v 45 min 700 or no band

Pick colony and colony PCR

Pour agar plate

take the 2 restriction endonuclease cuts from 37c n run gel (1.5 gel 65v 45 min) shd be ~5500bp and ~3500bp

 

11/9

PCR clean for 3 in 1 cut

Run gel for the dcas9 gfp sgrna no. 3 restriction endonuclease cut for dcas9 gfp sgrna (XbaI, XhoI), cut the 5500 band (65v 45 min) (big well) (all in)

Gel purification for the above cut gel (gm buffer 650ul)

Run gel for colony PCR yesterday (1.5 gel)

Set ligation mixture (restriction endonuclease cuts of 3 in 1 and mcy dcas9 gfp) 1:1

 

12/9

Heat inactivate ligation

2. Transform ligation (total dna 60ng)

3. Dilute overnight inoculation, 1 to 3

4. Set restriction endonuclease cut of 3 in 1 and sgrna dcas9 gfp

 

13/9

Measure od (growth curve) for modelling

16/9

Restriction endonuclease cuts for final product (XhoI, XbaI)

Pick colony for growth curve

 

17/9

Growth curve

Run gel for the restriction endonuclease cuts, PCR clean, start ligation

Transform ligation mixture

 

18/9

Transofrmation of final ligation products

Od measurements

 

19/9

pick colony (fat clone)

Transformation final product

 

20/9

Pick clone

PCR check band (Correct band at 12kbp)

Plasmid purification (Takara)

 

 

 

23/9

Pick colony of final product

 

24/9

Plasmid purification (Takara)

 

25/9

Nanodrop final product

 

26/9

Restriction endonuclease cut for final product (PstI)

 

27/9

Run gel for the restriction endonuclease cuts and final product 24 and 15.

All in for restriction endonuclease cuts,

5ul for final product 24 and 15

65v 50min

Nanodrop final product

 

30/9

Transformation

final product plasmid 15 17 24 29(BL21)

 

 

3/10

Observe green fluorescence by UV exposure

Inoculate

Observe green fluorescence again by UV exposure

All negative

 

4/10

Plasmid purification of final product

 

7/10

Transformation of final products into DH5a

 

8/10

Prepared 0.1mm nacl, 0.1mm hepes, 0.1mm vitb12, all not sterilized. Picked 10 colonies (final product) , inoculated in 20ml LB

 

9/10

Filtered Vitamin B12

Autoclave bold3n

Add to agar

 

10/10

Plasmid purification with ethanol percipitation

 

 

11/10

Measure Optical density (growth curve)

Transformation of Microcystis

Electroporation

Ligation by Peter

DNA purification

 

14/10

Transformation of previous ligation

 

15/10

Pick clones, inoculate i12

Standard solution

 

16/10

Pour plate with kanamycin resistance and chloramphenicol

Takara plasmid purification of insert 1 and insert 2

 

17/10

Peter ethanol precipitation of final product

Electroporation of Microcystis Aeruginosa UTEX 2388

Ligation mixture (cut with SpelI, xbaI)

Run gel for plasmid purification product of I2

Nanodrop I2

Anson adjust pH for BG11

Error: labelling error for plasmid purification

 

18/10

Pick insert 1 and 2 ,BL21 and NEB5a clones