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

 
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{{HK_SSC}}
 
{{HK_SSC}}
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<html>
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<head>
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<p>
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<meta http-equiv=Content-Type content="text/html; charset=utf-8">
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17 July
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</p>
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<meta name=Generator content="Microsoft Word 15">
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<p>
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<meta name=Originator content="Microsoft Word 15">
+
Transformation of dcas9 plasmid 2019 kit plate 5 well 22n.    
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+
</p>
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<p>
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<o:DocumentProperties>
+
- kanamycin resistance plate
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  <o:Author>(6L06) Lam Ngok</o:Author>
+
</p>
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  <o:LastAuthor>(4Y13) Young Hong Yui</o:LastAuthor>
+
<p>
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  <o:Revision>2</o:Revision>
+
- to E. coli NEB5a
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  <o:TotalTime>471</o:TotalTime>
+
</p>
−
  <o:Created>2019-10-22T02:04:00Z</o:Created>
+
<p>
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−
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</p>
−
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<p>
−
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19 July
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</p>
−
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<p>
−
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</p>
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</o:DocumentProperties>
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<p>
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Picked 6 colonies
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  <o:AllowPNG/>
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</p>
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<p>
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-grow overnight in LB
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</p>
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<link rel=colorSchemeMapping
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<p>
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+
Plasmid purification (Takara)  
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-colony 1 & 2 did not grow
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+
</p>
−
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+
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−
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-run agarose gel for 100v 30 mins
−
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</p>
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<p>
−
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-eluted DNA using 30ul elution buffer
−
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</p>
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-nanodrop the eluted DNA
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</p>
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<p>
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<p>
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22 July
−
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Plasmid prep 
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−
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<p>
−
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Run gel for dCas9
−
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+
</p>
−
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+
<p>
−
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Resuspend DNA
−
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23 July
−
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−
  <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
+
−
  Name="Table Grid 4"/>
+
−
  <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
+
−
  Name="Table Grid 5"/>
+
−
  <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
+
−
  Name="Table Grid 6"/>
+
−
  <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
+
−
  Name="Table Grid 7"/>
+
−
  <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
+
−
  Name="Table Grid 8"/>
+
−
  <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
+
−
  Name="Table List 1"/>
+
−
  <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
+
−
  Name="Table List 2"/>
+
−
  <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
+
−
  Name="Table List 3"/>
+
−
  <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
+
−
  Name="Table List 4"/>
+
−
  <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
+
−
  Name="Table List 5"/>
+
−
  <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
+
−
  Name="Table List 6"/>
+
−
  <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
+
−
  Name="Table List 7"/>
+
−
  <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
+
−
  Name="Table List 8"/>
+
−
  <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
+
−
  Name="Table 3D effects 1"/>
+
−
  <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
+
−
  Name="Table 3D effects 2"/>
+
−
  <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
+
−
  Name="Table 3D effects 3"/>
+
−
  <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
+
−
  Name="Table Contemporary"/>
+
−
  <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
+
−
  Name="Table Elegant"/>
+
−
  <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
+
−
  Name="Table Professional"/>
+
−
  <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
+
−
  Name="Table Subtle 1"/>
+
−
  <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
+
−
  Name="Table Subtle 2"/>
+
−
  <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
+
−
  Name="Table Web 1"/>
+
−
  <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
+
−
  Name="Table Web 2"/>
+
−
  <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
+
−
  Name="Table Web 3"/>
+
−
  <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
+
−
  Name="Balloon Text"/>
+
−
  <w:LsdException Locked="false" Priority="39" Name="Table Grid"/>
+
−
  <w:LsdException Locked="false" SemiHidden="true" UnhideWhenUsed="true"
+
−
  Name="Table Theme"/>
+
−
  <w:LsdException Locked="false" SemiHidden="true" Name="Placeholder Text"/>
+
−
  <w:LsdException Locked="false" Priority="1" QFormat="true" Name="No Spacing"/>
+
−
  <w:LsdException Locked="false" Priority="60" Name="Light Shading"/>
+
−
  <w:LsdException Locked="false" Priority="61" Name="Light List"/>
+
−
  <w:LsdException Locked="false" Priority="62" Name="Light Grid"/>
+
−
  <w:LsdException Locked="false" Priority="63" Name="Medium Shading 1"/>
+
−
  <w:LsdException Locked="false" Priority="64" Name="Medium Shading 2"/>
+
−
  <w:LsdException Locked="false" Priority="65" Name="Medium List 1"/>
+
−
  <w:LsdException Locked="false" Priority="66" Name="Medium List 2"/>
+
−
  <w:LsdException Locked="false" Priority="67" Name="Medium Grid 1"/>
+
−
  <w:LsdException Locked="false" Priority="68" Name="Medium Grid 2"/>
+
−
  <w:LsdException Locked="false" Priority="69" Name="Medium Grid 3"/>
+
−
  <w:LsdException Locked="false" Priority="70" Name="Dark List"/>
+
−
  <w:LsdException Locked="false" Priority="71" Name="Colorful Shading"/>
+
−
  <w:LsdException Locked="false" Priority="72" Name="Colorful List"/>
+
−
  <w:LsdException Locked="false" Priority="73" Name="Colorful Grid"/>
+
−
  <w:LsdException Locked="false" Priority="60" Name="Light Shading Accent 1"/>
+
−
  <w:LsdException Locked="false" Priority="61" Name="Light List Accent 1"/>
+
−
  <w:LsdException Locked="false" Priority="62" Name="Light Grid Accent 1"/>
+
−
  <w:LsdException Locked="false" Priority="63" Name="Medium Shading 1 Accent 1"/>
+
−
  <w:LsdException Locked="false" Priority="64" Name="Medium Shading 2 Accent 1"/>
+
−
  <w:LsdException Locked="false" Priority="65" Name="Medium List 1 Accent 1"/>
+
−
  <w:LsdException Locked="false" SemiHidden="true" Name="Revision"/>
+
−
  <w:LsdException Locked="false" Priority="34" QFormat="true"
+
−
  Name="List Paragraph"/>
+
−
  <w:LsdException Locked="false" Priority="29" QFormat="true" Name="Quote"/>
+
−
  <w:LsdException Locked="false" Priority="30" QFormat="true"
+
−
  Name="Intense Quote"/>
+
−
  <w:LsdException Locked="false" Priority="66" Name="Medium List 2 Accent 1"/>
+
−
  <w:LsdException Locked="false" Priority="67" Name="Medium Grid 1 Accent 1"/>
+
−
  <w:LsdException Locked="false" Priority="68" Name="Medium Grid 2 Accent 1"/>
+
−
  <w:LsdException Locked="false" Priority="69" Name="Medium Grid 3 Accent 1"/>
+
−
  <w:LsdException Locked="false" Priority="70" Name="Dark List Accent 1"/>
+
−
  <w:LsdException Locked="false" Priority="71" Name="Colorful Shading Accent 1"/>
+
−
  <w:LsdException Locked="false" Priority="72" Name="Colorful List Accent 1"/>
+
−
  <w:LsdException Locked="false" Priority="73" Name="Colorful Grid Accent 1"/>
+
−
  <w:LsdException Locked="false" Priority="60" Name="Light Shading Accent 2"/>
+
−
  <w:LsdException Locked="false" Priority="61" Name="Light List Accent 2"/>
+
−
  <w:LsdException Locked="false" Priority="62" Name="Light Grid Accent 2"/>
+
−
  <w:LsdException Locked="false" Priority="63" Name="Medium Shading 1 Accent 2"/>
+
−
  <w:LsdException Locked="false" Priority="64" Name="Medium Shading 2 Accent 2"/>
+
−
  <w:LsdException Locked="false" Priority="65" Name="Medium List 1 Accent 2"/>
+
−
  <w:LsdException Locked="false" Priority="66" Name="Medium List 2 Accent 2"/>
+
−
  <w:LsdException Locked="false" Priority="67" Name="Medium Grid 1 Accent 2"/>
+
−
  <w:LsdException Locked="false" Priority="68" Name="Medium Grid 2 Accent 2"/>
+
−
  <w:LsdException Locked="false" Priority="69" Name="Medium Grid 3 Accent 2"/>
+
−
  <w:LsdException Locked="false" Priority="70" Name="Dark List Accent 2"/>
+
−
  <w:LsdException Locked="false" Priority="71" Name="Colorful Shading Accent 2"/>
+
−
  <w:LsdException Locked="false" Priority="72" Name="Colorful List Accent 2"/>
+
−
  <w:LsdException Locked="false" Priority="73" Name="Colorful Grid Accent 2"/>
+
−
  <w:LsdException Locked="false" Priority="60" Name="Light Shading Accent 3"/>
+
−
  <w:LsdException Locked="false" Priority="61" Name="Light List Accent 3"/>
+
−
  <w:LsdException Locked="false" Priority="62" Name="Light Grid Accent 3"/>
+
−
  <w:LsdException Locked="false" Priority="63" Name="Medium Shading 1 Accent 3"/>
+
−
  <w:LsdException Locked="false" Priority="64" Name="Medium Shading 2 Accent 3"/>
+
−
  <w:LsdException Locked="false" Priority="65" Name="Medium List 1 Accent 3"/>
+
−
  <w:LsdException Locked="false" Priority="66" Name="Medium List 2 Accent 3"/>
+
−
  <w:LsdException Locked="false" Priority="67" Name="Medium Grid 1 Accent 3"/>
+
−
  <w:LsdException Locked="false" Priority="68" Name="Medium Grid 2 Accent 3"/>
+
−
  <w:LsdException Locked="false" Priority="69" Name="Medium Grid 3 Accent 3"/>
+
−
  <w:LsdException Locked="false" Priority="70" Name="Dark List Accent 3"/>
+
−
  <w:LsdException Locked="false" Priority="71" Name="Colorful Shading Accent 3"/>
+
−
  <w:LsdException Locked="false" Priority="72" Name="Colorful List Accent 3"/>
+
−
  <w:LsdException Locked="false" Priority="73" Name="Colorful Grid Accent 3"/>
+
−
  <w:LsdException Locked="false" Priority="60" Name="Light Shading Accent 4"/>
+
−
  <w:LsdException Locked="false" Priority="61" Name="Light List Accent 4"/>
+
−
  <w:LsdException Locked="false" Priority="62" Name="Light Grid Accent 4"/>
+
−
  <w:LsdException Locked="false" Priority="63" Name="Medium Shading 1 Accent 4"/>
+
−
  <w:LsdException Locked="false" Priority="64" Name="Medium Shading 2 Accent 4"/>
+
−
  <w:LsdException Locked="false" Priority="65" Name="Medium List 1 Accent 4"/>
+
−
  <w:LsdException Locked="false" Priority="66" Name="Medium List 2 Accent 4"/>
+
−
  <w:LsdException Locked="false" Priority="67" Name="Medium Grid 1 Accent 4"/>
+
−
  <w:LsdException Locked="false" Priority="68" Name="Medium Grid 2 Accent 4"/>
+
−
  <w:LsdException Locked="false" Priority="69" Name="Medium Grid 3 Accent 4"/>
+
−
  <w:LsdException Locked="false" Priority="70" Name="Dark List Accent 4"/>
+
−
  <w:LsdException Locked="false" Priority="71" Name="Colorful Shading Accent 4"/>
+
−
  <w:LsdException Locked="false" Priority="72" Name="Colorful List Accent 4"/>
+
−
  <w:LsdException Locked="false" Priority="73" Name="Colorful Grid Accent 4"/>
+
−
  <w:LsdException Locked="false" Priority="60" Name="Light Shading Accent 5"/>
+
−
  <w:LsdException Locked="false" Priority="61" Name="Light List Accent 5"/>
+
−
  <w:LsdException Locked="false" Priority="62" Name="Light Grid Accent 5"/>
+
−
  <w:LsdException Locked="false" Priority="63" Name="Medium Shading 1 Accent 5"/>
+
−
  <w:LsdException Locked="false" Priority="64" Name="Medium Shading 2 Accent 5"/>
+
−
  <w:LsdException Locked="false" Priority="65" Name="Medium List 1 Accent 5"/>
+
−
  <w:LsdException Locked="false" Priority="66" Name="Medium List 2 Accent 5"/>
+
−
  <w:LsdException Locked="false" Priority="67" Name="Medium Grid 1 Accent 5"/>
+
−
  <w:LsdException Locked="false" Priority="68" Name="Medium Grid 2 Accent 5"/>
+
−
  <w:LsdException Locked="false" Priority="69" Name="Medium Grid 3 Accent 5"/>
+
−
  <w:LsdException Locked="false" Priority="70" Name="Dark List Accent 5"/>
+
−
  <w:LsdException Locked="false" Priority="71" Name="Colorful Shading Accent 5"/>
+
−
  <w:LsdException Locked="false" Priority="72" Name="Colorful List Accent 5"/>
+
−
  <w:LsdException Locked="false" Priority="73" Name="Colorful Grid Accent 5"/>
+
−
  <w:LsdException Locked="false" Priority="60" Name="Light Shading Accent 6"/>
+
−
  <w:LsdException Locked="false" Priority="61" Name="Light List Accent 6"/>
+
−
  <w:LsdException Locked="false" Priority="62" Name="Light Grid Accent 6"/>
+
−
  <w:LsdException Locked="false" Priority="63" Name="Medium Shading 1 Accent 6"/>
+
−
  <w:LsdException Locked="false" Priority="64" Name="Medium Shading 2 Accent 6"/>
+
−
  <w:LsdException Locked="false" Priority="65" Name="Medium List 1 Accent 6"/>
+
−
  <w:LsdException Locked="false" Priority="66" Name="Medium List 2 Accent 6"/>
+
−
  <w:LsdException Locked="false" Priority="67" Name="Medium Grid 1 Accent 6"/>
+
−
  <w:LsdException Locked="false" Priority="68" Name="Medium Grid 2 Accent 6"/>
+
−
  <w:LsdException Locked="false" Priority="69" Name="Medium Grid 3 Accent 6"/>
+
−
  <w:LsdException Locked="false" Priority="70" Name="Dark List Accent 6"/>
+
−
  <w:LsdException Locked="false" Priority="71" Name="Colorful Shading Accent 6"/>
+
−
  <w:LsdException Locked="false" Priority="72" Name="Colorful List Accent 6"/>
+
−
  <w:LsdException Locked="false" Priority="73" Name="Colorful Grid Accent 6"/>
+
−
  <w:LsdException Locked="false" Priority="19" QFormat="true"
+
−
  Name="Subtle Emphasis"/>
+
−
  <w:LsdException Locked="false" Priority="21" QFormat="true"
+
−
  Name="Intense Emphasis"/>
+
−
  <w:LsdException Locked="false" Priority="31" QFormat="true"
+
−
  Name="Subtle Reference"/>
+
−
  <w:LsdException Locked="false" Priority="32" QFormat="true"
+
−
  Name="Intense Reference"/>
+
−
  <w:LsdException Locked="false" Priority="33" QFormat="true" Name="Book Title"/>
+
−
  <w:LsdException Locked="false" Priority="37" SemiHidden="true"
+
−
  UnhideWhenUsed="true" Name="Bibliography"/>
+
−
  <w:LsdException Locked="false" Priority="39" SemiHidden="true"
+
−
  UnhideWhenUsed="true" QFormat="true" Name="TOC Heading"/>
+
−
  <w:LsdException Locked="false" Priority="41" Name="Plain Table 1"/>
+
−
  <w:LsdException Locked="false" Priority="42" Name="Plain Table 2"/>
+
−
  <w:LsdException Locked="false" Priority="43" Name="Plain Table 3"/>
+
−
  <w:LsdException Locked="false" Priority="44" Name="Plain Table 4"/>
+
−
  <w:LsdException Locked="false" Priority="45" Name="Plain Table 5"/>
+
−
  <w:LsdException Locked="false" Priority="40" Name="Grid Table Light"/>
+
−
  <w:LsdException Locked="false" Priority="46" Name="Grid Table 1 Light"/>
+
−
  <w:LsdException Locked="false" Priority="47" Name="Grid Table 2"/>
+
−
  <w:LsdException Locked="false" Priority="48" Name="Grid Table 3"/>
+
−
  <w:LsdException Locked="false" Priority="49" Name="Grid Table 4"/>
+
−
  <w:LsdException Locked="false" Priority="50" Name="Grid Table 5 Dark"/>
+
−
  <w:LsdException Locked="false" Priority="51" Name="Grid Table 6 Colorful"/>
+
−
  <w:LsdException Locked="false" Priority="52" Name="Grid Table 7 Colorful"/>
+
−
  <w:LsdException Locked="false" Priority="46"
+
−
  Name="Grid Table 1 Light Accent 1"/>
+
−
  <w:LsdException Locked="false" Priority="47" Name="Grid Table 2 Accent 1"/>
+
−
  <w:LsdException Locked="false" Priority="48" Name="Grid Table 3 Accent 1"/>
+
−
  <w:LsdException Locked="false" Priority="49" Name="Grid Table 4 Accent 1"/>
+
−
  <w:LsdException Locked="false" Priority="50" Name="Grid Table 5 Dark Accent 1"/>
+
−
  <w:LsdException Locked="false" Priority="51"
+
−
  Name="Grid Table 6 Colorful Accent 1"/>
+
−
  <w:LsdException Locked="false" Priority="52"
+
−
  Name="Grid Table 7 Colorful Accent 1"/>
+
−
  <w:LsdException Locked="false" Priority="46"
+
−
  Name="Grid Table 1 Light Accent 2"/>
+
−
  <w:LsdException Locked="false" Priority="47" Name="Grid Table 2 Accent 2"/>
+
−
  <w:LsdException Locked="false" Priority="48" Name="Grid Table 3 Accent 2"/>
+
−
  <w:LsdException Locked="false" Priority="49" Name="Grid Table 4 Accent 2"/>
+
−
  <w:LsdException Locked="false" Priority="50" Name="Grid Table 5 Dark Accent 2"/>
+
−
  <w:LsdException Locked="false" Priority="51"
+
−
  Name="Grid Table 6 Colorful Accent 2"/>
+
−
  <w:LsdException Locked="false" Priority="52"
+
−
  Name="Grid Table 7 Colorful Accent 2"/>
+
−
  <w:LsdException Locked="false" Priority="46"
+
−
  Name="Grid Table 1 Light Accent 3"/>
+
−
  <w:LsdException Locked="false" Priority="47" Name="Grid Table 2 Accent 3"/>
+
−
  <w:LsdException Locked="false" Priority="48" Name="Grid Table 3 Accent 3"/>
+
−
  <w:LsdException Locked="false" Priority="49" Name="Grid Table 4 Accent 3"/>
+
−
  <w:LsdException Locked="false" Priority="50" Name="Grid Table 5 Dark Accent 3"/>
+
−
  <w:LsdException Locked="false" Priority="51"
+
−
  Name="Grid Table 6 Colorful Accent 3"/>
+
−
  <w:LsdException Locked="false" Priority="52"
+
−
  Name="Grid Table 7 Colorful Accent 3"/>
+
−
  <w:LsdException Locked="false" Priority="46"
+
−
  Name="Grid Table 1 Light Accent 4"/>
+
−
  <w:LsdException Locked="false" Priority="47" Name="Grid Table 2 Accent 4"/>
+
−
  <w:LsdException Locked="false" Priority="48" Name="Grid Table 3 Accent 4"/>
+
−
  <w:LsdException Locked="false" Priority="49" Name="Grid Table 4 Accent 4"/>
+
−
  <w:LsdException Locked="false" Priority="50" Name="Grid Table 5 Dark Accent 4"/>
+
−
  <w:LsdException Locked="false" Priority="51"
+
−
  Name="Grid Table 6 Colorful Accent 4"/>
+
−
  <w:LsdException Locked="false" Priority="52"
+
−
  Name="Grid Table 7 Colorful Accent 4"/>
+
−
  <w:LsdException Locked="false" Priority="46"
+
−
  Name="Grid Table 1 Light Accent 5"/>
+
−
  <w:LsdException Locked="false" Priority="47" Name="Grid Table 2 Accent 5"/>
+
−
  <w:LsdException Locked="false" Priority="48" Name="Grid Table 3 Accent 5"/>
+
−
  <w:LsdException Locked="false" Priority="49" Name="Grid Table 4 Accent 5"/>
+
−
  <w:LsdException Locked="false" Priority="50" Name="Grid Table 5 Dark Accent 5"/>
+
−
  <w:LsdException Locked="false" Priority="51"
+
−
  Name="Grid Table 6 Colorful Accent 5"/>
+
−
  <w:LsdException Locked="false" Priority="52"
+
−
  Name="Grid Table 7 Colorful Accent 5"/>
+
−
  <w:LsdException Locked="false" Priority="46"
+
−
  Name="Grid Table 1 Light Accent 6"/>
+
−
  <w:LsdException Locked="false" Priority="47" Name="Grid Table 2 Accent 6"/>
+
−
  <w:LsdException Locked="false" Priority="48" Name="Grid Table 3 Accent 6"/>
+
−
  <w:LsdException Locked="false" Priority="49" Name="Grid Table 4 Accent 6"/>
+
−
  <w:LsdException Locked="false" Priority="50" Name="Grid Table 5 Dark Accent 6"/>
+
−
  <w:LsdException Locked="false" Priority="51"
+
−
  Name="Grid Table 6 Colorful Accent 6"/>
+
−
  <w:LsdException Locked="false" Priority="52"
+
−
  Name="Grid Table 7 Colorful Accent 6"/>
+
−
  <w:LsdException Locked="false" Priority="46" Name="List Table 1 Light"/>
+
−
  <w:LsdException Locked="false" Priority="47" Name="List Table 2"/>
+
−
  <w:LsdException Locked="false" Priority="48" Name="List Table 3"/>
+
−
  <w:LsdException Locked="false" Priority="49" Name="List Table 4"/>
+
−
  <w:LsdException Locked="false" Priority="50" Name="List Table 5 Dark"/>
+
−
  <w:LsdException Locked="false" Priority="51" Name="List Table 6 Colorful"/>
+
−
  <w:LsdException Locked="false" Priority="52" Name="List Table 7 Colorful"/>
+
−
  <w:LsdException Locked="false" Priority="46"
+
−
  Name="List Table 1 Light Accent 1"/>
+
−
  <w:LsdException Locked="false" Priority="47" Name="List Table 2 Accent 1"/>
+
−
  <w:LsdException Locked="false" Priority="48" Name="List Table 3 Accent 1"/>
+
−
  <w:LsdException Locked="false" Priority="49" Name="List Table 4 Accent 1"/>
+
−
  <w:LsdException Locked="false" Priority="50" Name="List Table 5 Dark Accent 1"/>
+
−
  <w:LsdException Locked="false" Priority="51"
+
−
  Name="List Table 6 Colorful Accent 1"/>
+
−
  <w:LsdException Locked="false" Priority="52"
+
−
  Name="List Table 7 Colorful Accent 1"/>
+
−
  <w:LsdException Locked="false" Priority="46"
+
−
  Name="List Table 1 Light Accent 2"/>
+
−
  <w:LsdException Locked="false" Priority="47" Name="List Table 2 Accent 2"/>
+
−
  <w:LsdException Locked="false" Priority="48" Name="List Table 3 Accent 2"/>
+
−
  <w:LsdException Locked="false" Priority="49" Name="List Table 4 Accent 2"/>
+
−
  <w:LsdException Locked="false" Priority="50" Name="List Table 5 Dark Accent 2"/>
+
−
  <w:LsdException Locked="false" Priority="51"
+
−
  Name="List Table 6 Colorful Accent 2"/>
+
−
  <w:LsdException Locked="false" Priority="52"
+
−
  Name="List Table 7 Colorful Accent 2"/>
+
−
  <w:LsdException Locked="false" Priority="46"
+
−
  Name="List Table 1 Light Accent 3"/>
+
−
  <w:LsdException Locked="false" Priority="47" Name="List Table 2 Accent 3"/>
+
−
  <w:LsdException Locked="false" Priority="48" Name="List Table 3 Accent 3"/>
+
−
  <w:LsdException Locked="false" Priority="49" Name="List Table 4 Accent 3"/>
+
−
  <w:LsdException Locked="false" Priority="50" Name="List Table 5 Dark Accent 3"/>
+
−
  <w:LsdException Locked="false" Priority="51"
+
−
  Name="List Table 6 Colorful Accent 3"/>
+
−
  <w:LsdException Locked="false" Priority="52"
+
−
  Name="List Table 7 Colorful Accent 3"/>
+
−
  <w:LsdException Locked="false" Priority="46"
+
−
  Name="List Table 1 Light Accent 4"/>
+
−
  <w:LsdException Locked="false" Priority="47" Name="List Table 2 Accent 4"/>
+
−
  <w:LsdException Locked="false" Priority="48" Name="List Table 3 Accent 4"/>
+
−
  <w:LsdException Locked="false" Priority="49" Name="List Table 4 Accent 4"/>
+
−
  <w:LsdException Locked="false" Priority="50" Name="List Table 5 Dark Accent 4"/>
+
−
  <w:LsdException Locked="false" Priority="51"
+
−
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+
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+
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+
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+
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+
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mso-hansi-theme-font:minor-latin;
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mso-bidi-theme-font:minor-bidi;}
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<![endif]--><!--[if gte mso 9]><xml>
+
−
<o:shapedefaults v:ext="edit" spidmax="2050"/>
+
−
</xml><![endif]--><!--[if gte mso 9]><xml>
+
−
<o:shapelayout v:ext="edit">
+
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  <o:idmap v:ext="edit" data="1"/>
+
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</o:shapelayout></xml><![endif]-->
+
−
</head>
+
  
−
<body lang=EN-HK style='tab-interval:36.0pt'>
+
<li>Assembling Fragment 1+2+3
 +
</li>
 +
</ol>
 +
<p>
 +
-restriction endonuclease cut + ligation
 +
</p>
 +
<p>
 +
NheI and BssSI-v2
 +
</p>
 +
<p>
 +
 +
</p>
 +
<ol>
  
−
<div class=WordSection1>
+
<li>Dcas9 preparation clean
  
−
<div style='mso-element:para-border-div;border:none;border-right:solid windowtext 1.0pt;
+
<li>Run gel  NEB5α
−
mso-border-right-alt:solid windowtext .5pt;padding:0cm 0cm 0cm 0cm'>
+
</li>
 +
</ol>
 +
<p>
 +
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
Transformed produce to E.coli neb5
 +
</p>
 +
<p>
 +
 +
</p>
 +
<ol>
  
−
<p class=MsoNormal style='line-height:150%;border:none;mso-border-right-alt:
+
<li>Cloned GFP to dcas9
−
solid windowtext .5pt;padding:0cm;mso-padding-alt:0cm 0cm 0cm 0cm'><b
+
</li>
−
style='mso-bidi-font-weight:normal'><span lang=EN-US style='font-family:"Times New Roman",serif;
+
</ol>
−
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>17 <span
+
<p>
−
style='mso-bidi-font-weight:bold'>July</span></span></b><b><span lang=EN-US
+
-restriction endonuclease cut + ligation
−
style='font-family:Times;mso-bidi-font-family:"Times New Roman";mso-ansi-language:
+
</p>
−
EN-US'><span style='mso-tab-count:6'>                                                                        </span></span></b><b
+
<p>
−
style='mso-bidi-font-weight:normal'><span lang=EN-US style='font-family:"Times New Roman",serif;
+
EcorI NsiI
−
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'><o:p></o:p></span></b></p>
+
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
Transformed product to E.coli neb5
 +
</p>
 +
<p>
 +
 +
</p>
 +
<ol>
  
−
</div>
+
<li>Transformed puc19 from stock (for cloning sgrna later)
 +
</li>
 +
</ol>
 +
<p>
 +
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
1 Aug
 +
</p>
 +
<p>
 +
Cyanobacteria arrived, cultured in 15x4 ml medium
 +
</p>
 +
<p>
 +
GFP-dcas9 plasmid ligation (all failed)
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
2 Aug
 +
</p>
 +
<p>
 +
Colony PCR for l2, 3, 4
 +
</p>
 +
<p>
 +
PCR Fragment 1, 2, 3
 +
</p>
 +
<p>
 +
Plasmid purification
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
6 Aug
 +
</p>
 +
<p>
 +
-colony PCR 
 +
</p>
 +
<p>
 +
-standard q5 PCR preperation for gibson 
 +
</p>
 +
<p>
 +
-gibson
 +
</p>
 +
<p>
 +
- gfp+ dcas9 plasmid again as l3
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
 
 +
</p>
 +
<p>
 +
7 Aug
 +
</p>
 +
<p>
 +
-gel purification after 75v 45min 0.7 gel 
 +
</p>
 +
<p>
 +
- gibson assembly 
 +
</p>
 +
<p>
 +
- run gel for overnight sgrna PCR product 
 +
</p>
 +
<p>
 +
- restriction endonuclease cut(PstI, HindIII)(2hrs incubation)+ligation for sgrna and petblue 
 +
</p>
 +
<p>
 +
-transformed 3in1, petblue sgrna and L3 into NEB5a
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
8 Aug
 +
</p>
 +
<p>
 +
Colony PCR for l3 
 +
</p>
 +
<p>
 +
Run gel for 3in1 (1 smear), 
 +
</p>
 +
<p>
 +
Pick clone
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
9 August
 +
</p>
 +
<ol>
  
−
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
+
<li>Checked plates for sgrna pet blue mcyi
−
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Transformation
+
</li>
−
of dcas9 plasmid 2019 kit plate 5 well 22N<span style='mso-spacerun:yes'>  
+
</ol>
−
</span><o:p></o:p></span></p>
+
<p>
 +
Sgrna pet blue mcyI2
 +
</p>
 +
<p>
 +
Sgrna pet blue mcyb 2
 +
</p>
 +
<p>
 +
3 in 1
 +
</p>
 +
<p>
 +
Gib 2.1
 +
</p>
 +
<p>
 +
Gib 2.2
 +
</p>
 +
<p>
 +
-all no colony.
 +
</p>
 +
<ol>
  
−
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
+
<li>Conducted PCR purification of overnight (14h) restriction endonuclease cut of dcas9 gfp (EcoRI, NsiI)
−
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>to E. coli
+
</li>
−
NEB5</span><span lang=EN-US style='font-family:Symbol;mso-fareast-font-family:
+
</ol>
−
Symbol;mso-bidi-font-family:Symbol;mso-ansi-language:EN-US'>a</span><span
+
<p>
−
lang=EN-US style='font-family:"Times New Roman",serif;mso-fareast-font-family:
+
-50 rxn mixture
−
"Times New Roman";mso-ansi-language:EN-US'> using kanamycin resistance plate<o:p></o:p></span></p>
+
</p>
 +
<ol>
  
−
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
+
<li>Conducted ligation
−
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'><o:p>&nbsp;</o:p></span></p>
+
</li>
 +
</ol>
 +
<p>
 +
1:3 & 1:5
 +
</p>
 +
<p>
 +
Vector 250ng
 +
</p>
 +
<p>
 +
Both 20ul rxn,  
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
-rm temp for 10 min
 +
</p>
 +
<p>
 +
heat inactive at 65c for 10 min
 +
</p>
 +
<p>
 +
on ice 90min
 +
</p>
 +
<p>
 +
-then add ligase again
 +
</p>
 +
<p>
 +
ligate over weekend.
 +
</p>
 +
<p>
 +
 +
</p>
 +
<ol>
  
−
<p class=MsoNormal><b style='mso-bidi-font-weight:normal'><span lang=EN-US
+
<li>Amplified Fragment1 + 2 + 3
−
style='font-family:"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";
+
</li>
−
mso-ansi-language:EN-US'>19 <span style='mso-bidi-font-weight:bold'>July</span><o:p></o:p></span></b></p>
+
</ol>
 +
<p>
 +
-sgrna I, sgrna B, gfp
 +
</p>
 +
<p>
 +
For gibson
 +
</p>
 +
<p>
 +
Fragment 1 2 3 gfp, 65c
 +
</p>
 +
<p>
 +
Sgrnas 55c
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
All 50 ul rxn
 +
</p>
 +
<p>
 +
 +
</p>
 +
<ol>
  
−
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
+
<li>Weight powder, for soc (800ml no glucose/ mgxx) (no yellow)
−
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Picked 6
+
</li>
−
colonies<o:p></o:p></span></p>
+
</ol>
 +
<p>
 +
-LB agar (have yellow) 1800ul
 +
</p>
 +
<p>
 +
Glucose 9g
 +
</p>
 +
<p>
 +
 +
</p>
 +
<ol>
  
−
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
+
<li>Run gel for all 6 Fragments (all in)
−
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>grow
+
</li>
−
overnight in LB<o:p></o:p></span></p>
+
</ol>
 +
<p>
 +
-75v 45 mins
 +
</p>
 +
<p>
 +
Ladder use 1kb & 50bp
 +
</p>
 +
<p>
 +
Loading dye 6x
 +
</p>
 +
<p>
 +
 +
</p>
 +
<ol>
  
−
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
+
<li>Gel purification -elude using 30ul elution b.
−
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Plasmid
+
</li>
−
purification (Takara)<o:p></o:p></span></p>
+
</ol>
 +
<p>
 +
 +
</p>
 +
<p>
 +
12 August
 +
</p>
 +
<p>
 +
 +
</p>
 +
<ol>
  
−
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
+
<li>Colony PCR for those without bands reaction 
−
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>colony 1
+
</li>
−
&amp; 2 did not grow<o:p></o:p></span></p>
+
</ol>
 +
<p>
 +
-picked colonies from master plate
 +
</p>
 +
<p>
 +
 +
</p>
 +
<ol>
  
−
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
+
<li>Performed gibson(gib3) & nebuilder
−
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>run agarose gel
+
−
for 100v 30 mins<o:p></o:p></span></p>
+
  
−
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
+
<li>Scaled down to 10ml rxn
−
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>eluted DNA
+
−
using 30ul elution buffer<o:p></o:p></span></p>
+
  
−
<p class=MsoNormal style='tab-stops:91.55pt'><span lang=EN-US style='font-family:
+
<li>Concentration acc to following nanodrop
−
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
+
</li>
−
EN-US'>nanodrop the eluted DNA</span><span lang=EN-US style='font-family:Times;
+
</ol>
−
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:
+
-incubation 1h
−
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
+
</p>
−
EN-US'><o:p></o:p></span></p>
+
<p>
 +
 +
</p>
 +
<ol>
  
−
<p class=MsoNormal><b style='mso-bidi-font-weight:normal'><span lang=EN-US
+
<li>PCR for xgib 1 (Fragment1) gib1 (Fragment1)
−
style='font-family:"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";
+
</li>
−
mso-ansi-language:EN-US'><o:p>&nbsp;</o:p></span></b></p>
+
</ol>
 +
<p>
 +
            xgib 2 (Fragment2) gib2 (Fragment2)
 +
</p>
 +
<p>
 +
xgib 3 (Fragment3) gib3 (Fragment3)
 +
</p>
 +
<p>
 +
 +
</p>
 +
<ol>
  
−
<p class=MsoNormal><b style='mso-bidi-font-weight:normal'><span lang=EN-US
+
<li>Colony PCR no.24 correct band
−
style='font-family:"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";
+
</li>
−
mso-ansi-language:EN-US'>22 <span style='mso-bidi-font-weight:bold'>July</span><o:p></o:p></span></b></p>
+
</ol>
 +
<p>
 +
(dcas9 gfp) L3
 +
</p>
 +
<p>
 +
band size 700-800 bp
 +
</p>
 +
<p>
 +
            inoculated 24 Hours (picked from master plate)
 +
</p>
 +
<p>
 +
 +
</p>
 +
<ol>
  
−
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
+
<li>Transformed gibson 1 gibson 2 gibson 3.2  nebuilder (1)
−
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Plasmid prep
+
</li>
−
<o:p></o:p></span></p>
+
</ol>
 +
<p>
 +
+ve control  petblue nebuilder (2)
 +
</p>
 +
<p>
 +
      dcas9 gfp 1:3
 +
</p>
 +
<p>
 +
      dcas9 gfp 1:5
 +
</p>
 +
<p>
 +
          gibson 3.1
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
13 August
 +
</p>
 +
<p>
 +
 +
</p>
 +
<ol>
  
−
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
+
<li>Run gel for previous PCR 
−
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Run gel for dCas9<o:p></o:p></span></p>
+
</li>
 +
</ol>
 +
<p>
 +
 +
</p>
 +
<ol>
  
−
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
+
<li>Checked plates from 12/8
−
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Resuspend DNA<o:p></o:p></span></p>
+
</li>
 +
</ol>
 +
<p>
 +
Colony: petblue +ve
 +
</p>
 +
<p>
 +
dCas9 gfp 1:3
 +
</p>
 +
<p>
 +
dCas9 gfp 1:5
 +
</p>
 +
<p>
 +
 +
</p>
 +
<ol>
  
−
<p class=MsoNormal><b style='mso-bidi-font-weight:normal'><span lang=EN-US
+
<li>Plasmid purification l3
−
style='font-family:"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";
+
</li>
−
mso-ansi-language:EN-US'><o:p>&nbsp;</o:p></span></b></p>
+
</ol>
 +
<p>
 +
dCas9-GFP 24
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
Re cut for Fragment1-3 (gibson assembly)
 +
</p>
 +
<p>
 +
McyI, Mcyb
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
14 August
 +
</p>
 +
<p>
 +
 +
</p>
 +
<ol>
  
−
<p class=MsoNormal><b style='mso-bidi-font-weight:normal'><span lang=EN-US
+
<li>Run gel for xgib Fragment 1
−
style='font-family:"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";
+
</li>
−
mso-ansi-language:EN-US'>23 <span style='mso-bidi-font-weight:bold'>July</span><o:p></o:p></span></b></p>
+
</ol>
 +
<p>
 +
-no bands at all
 +
</p>
 +
<p>
 +
Possibly due to pipetting errors.
 +
</p>
 +
<p>
 +
 +
</p>
 +
<ol>
  
−
<div style='mso-element:para-border-div;border:none;border-right:solid windowtext 1.0pt;
+
<li>Started 10ul reaction using gibson concentration according to following gel. (pic gib4)
−
mso-border-right-alt:solid windowtext .5pt;padding:0cm 4.0pt 0cm 0cm'>
+
</li>
 +
</ol>
 +
<p>
 +
 +
</p>
 +
<p>
 +
Reaction: Fragment3: 50ng
 +
</p>
 +
<p>
 +
    Fragment2: 38.73ng
 +
</p>
 +
<p>
 +
    Fragment1: 29.26ng
 +
</p>
 +
<p>
 +
                nebuilder: 5ul
 +
</p>
 +
<p>
 +
h2o
 +
</p>
 +
<p>
 +
 +
</p>
 +
<ol>
  
−
<p class=MsoNormal style='border:none;mso-border-right-alt:solid windowtext .5pt;
+
<li>Set up PCR reaction for xgib Fragment2
−
padding:0cm;mso-padding-alt:0cm 4.0pt 0cm 0cm'><span lang=EN-US
+
</li>
−
style='font-family:"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";
+
</ol>
−
mso-ansi-language:EN-US'>Assembling Fragment 1+2+3</span><span lang=EN-US
+
<p>
−
style='font-family:Times;mso-bidi-font-family:"Times New Roman";mso-ansi-language:
+
Xgib Fragment3
−
EN-US'><o:p></o:p></span></p>
+
</p>
 +
<p>
 +
 +
</p>
  
−
</div>
+
<table>
−
 
+
   <tr>
−
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
+
  <td>98c
−
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>restriction
+
  </td>
−
endonuclease cut + ligation<o:p></o:p></span></p>
+
  <td>98c
−
 
+
  </td>
−
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
+
  <td>66c
−
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Using <span
+
  </td>
−
class=SpellE>NheI</span> and BssSI-v2<o:p></o:p></span></p>
+
  <td>72c
−
 
+
  </td>
−
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
+
  <td>72c
−
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Dcas9
+
  </td>
−
preparation clean</span><span lang=EN-US style='font-family:Times;mso-bidi-font-family:
+
  <td>4c
−
"Times New Roman";mso-ansi-language:EN-US'><o:p></o:p></span></p>
+
  </td>
−
 
+
   </tr>
−
<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'>Run <span
+
  <td>30sec  
−
class=GramE>gel <span style='mso-spacerun:yes'> </span>dCas</span>9</span><span
+
  </td>
−
style='font-size:11.0pt;font-family:Times;mso-fareast-font-family:"Times New Roman";
+
  <td>5s
−
mso-bidi-font-family:"Times New Roman";color:#222222'><o:p></o:p></span></p>
+
  </td>
−
 
+
  <td>15s
−
<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'>Transformed
+
  <td>1.5min  
−
produce to <span class=GramE>E.coli</span> neb5</span><span lang=EN-US
+
  </td>
−
style='font-family:Symbol;mso-fareast-font-family:"Times New Roman";mso-bidi-font-family:
+
  <td>2min  
−
"Times New Roman";mso-ansi-language:EN-US'>a<o:p></o:p></span></p>
+
  </td>
−
 
+
  <td>  
−
<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'>Cloned GFP
+
   </tr>
−
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>
+
−
 
+
−
<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 + ligation<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'>Using <span
+
−
class=SpellE>EcorI</span> <span class=SpellE>NsiI</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'>Transformed
+
−
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>
+
−
 
+
−
<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 puc19
+
−
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>
+
−
 
+
−
<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><span lang=EN-US style='font-family:"Times New Roman",serif;
+
−
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>1 Aug<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'>Cyanobacteria
+
−
arrived, cultured in 15x4 ml medium<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'>GFP-dcas9
+
−
plasmid ligation (all failed)<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><span lang=EN-US style='font-family:"Times New Roman",serif;
+
−
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>2 Aug<o:p></o:p></span></b></p>
+
−
 
+
−
<p class=MsoNormal style='line-height:107%'><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 L2, 3, 4<o:p></o:p></span></p>
+
−
 
+
−
<p class=MsoNormal style='line-height:107%'><span lang=EN-US style='font-family:
+
−
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
+
−
EN-US'>PCR Fragment 1, 2, 3<o:p></o:p></span></p>
+
−
 
+
−
<p class=MsoNormal style='line-height:107%'><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<o:p></o:p></span></p>
+
−
 
+
−
<p class=MsoNormal style='line-height:107%'><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'>6 Aug<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 <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'>standard q5 PCR
+
−
<span class=SpellE>preperation</span> for <span class=SpellE>gibson</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'>Gibson assembly<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'>GFP+ dCas9
+
−
plasmid again as L3 Failed<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'><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'>7 Aug<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 after 75v 45min 0.7 gel <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'>gibson</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'> assembly <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 overnight
+
−
<span class=SpellE>sgrna</span> PCR product <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 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>
+
−
 
+
−
<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
+
−
3in1, <span class=SpellE>PETblue</span> sgRNA and L3 into 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'><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'>8 Aug<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
+
−
for l3 <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
+
−
3in1 (1 smear), <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'>Pick clone<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><span lang=EN-US style='font-family:"Times New Roman",serif;
+
−
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>9 August</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>
+
−
 
+
−
<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 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>
+
−
 
+
−
<p class=MsoNormal style='margin-left:72.0pt'><span lang=EN-US
+
−
style='font-family:Times;mso-bidi-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 New Roman",serif;mso-fareast-font-family:"Times New Roman";
+
−
mso-ansi-language:EN-US'>sgRNA pet blue <span class=SpellE><span class=GramE>McyI</span></span><span
+
−
class=GramE>(</span>no colony)<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>sgRNA pet blue <span
+
−
class=SpellE>Mcyb</span> (no colony)<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>3 in 1(no colony)<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>Gib 2.1(no
+
−
colony)<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>Gib 2.2(no colony)<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'>Conducted PCR
+
−
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>
+
−
 
+
−
<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'>50 </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'>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>
+
−
 
+
−
<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'>Conducted
+
−
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'>1:3 &amp;
+
−
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'>Vector 250ng<o:p></o:p></span></p>
+
−
 
+
−
<p class=MsoNormal><span lang=EN-US style='font-family:Times;mso-fareast-font-family:
+
−
"Times New Roman";mso-bidi-font-family:"Times New Roman";mso-ansi-language:
+
−
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>
+
−
 
+
−
<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'>Placed in room
+
−
temperature for 10 min<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'>heat
+
−
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>
+
−
 
+
−
<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'>on ice 90min<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'>then add
+
−
ligase again<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'>ligate over
+
−
weekend.<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 Fragment1
+
−
+ 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=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'>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>McyI</span>, sgRNA
+
−
<span class=SpellE>McyB</span>, GFP<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'>Fragment 1 2
+
−
3 GFP, 65c<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'>sgRNA 55c<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 50 </span><span
+
−
class=SpellE><span style='font-family:"Arial",sans-serif;mso-fareast-font-family:
+
−
"Times New Roman";color:#222222;background:white'>μL</span></span><span
+
−
style='font-family:"Times New Roman",serif;mso-fareast-font-family:"Times New Roman"'>
+
−
</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'>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 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>
 
−
 
−
<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'>13/9<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'>Measure od (growth curve) for modelling<o:p></o:p></span></p>
 
−
 
−
<p class=MsoNormal style='tab-stops:5.0cm'><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=MsoNormal style='tab-stops:5.0cm'><span lang=EN-US style='font-family:
 
−
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
 
−
EN-US'>16/9<o:p></o:p></span></p>
 
−
 
−
<p class=MsoNormal style='tab-stops:5.0cm'><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 cuts for final product (<span class=SpellE>XhoI</span>,
 
−
<span class=SpellE>XbaI</span>)<o:p></o:p></span></p>
 
−
 
−
<p class=MsoNormal style='tab-stops:5.0cm'><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 for growth curve<o:p></o:p></span></p>
 
−
 
−
<p class=MsoNormal style='tab-stops:5.0cm'><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:5.0cm'><span lang=EN-US style='font-family:
 
−
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
 
−
EN-US'>17/9<o:p></o:p></span></p>
 
−
 
−
<p class=MsoNormal style='tab-stops:5.0cm'><span lang=EN-US style='font-family:
 
−
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
 
−
EN-US'>Growth curve<o:p></o:p></span></p>
 
−
 
−
<p class=MsoNormal style='tab-stops:5.0cm'><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 restriction endonuclease cuts, PCR clean, start ligation<o:p></o:p></span></p>
 
−
 
−
<p class=MsoNormal style='tab-stops:5.0cm'><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 mixture<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'>18/9<o:p></o:p></span></p>
 
−
 
−
<p class=MsoNormal style='tab-stops:283.0pt'><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'>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>
 
−
 
−
<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'>Od measurements<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'>19/9<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'>pick colony (fat clone)<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 final product<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'>20/9<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'>Pick clone<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 check band (Correct band at 12kbp)<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 (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'><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'><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'><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'>23/9<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'>Pick colony of final product<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";color:black;
 
−
mso-themecolor:text1;mso-ansi-language:EN-US'>24/9<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 (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'><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'>25/9 <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'>Nanodrop final product<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:black;
 
−
mso-themecolor:text1;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";color:black;
 
−
mso-themecolor:text1;mso-ansi-language:EN-US'>26/9<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'>Restriction
 
−
endonuclease cut for final product (<span class=SpellE>PstI</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 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'>27/9<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 restriction endonuclease cuts and final product 24 and
 
−
15. <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'>All in for restriction endonuclease cuts, <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'>5ul for final product 24 and 15<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'>65v 50min<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'>Nanodrop final product<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'>30/9<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<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>final product plasmid 15 17 24
 
−
29(BL21)<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'><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'>3/10<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'>Observe green fluorescence by UV exposure<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<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'>Observe green fluorescence again by UV exposure<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 negative
 
−
</span><span lang=EN-US style='font-family:Times;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-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";color:black;
 
−
mso-themecolor:text1;mso-ansi-language:EN-US'>4/10<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:black;
 
−
mso-themecolor:text1;mso-ansi-language:EN-US'>Plasmid purification of final
 
−
product<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";color:black;
 
−
mso-themecolor:text1;mso-ansi-language:EN-US'>7/10<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:black;
 
−
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>
 
−
 
−
<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'>8/10<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'>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>
 
−
 
−
<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><span lang=EN-US style='font-family:"Times New Roman",serif;
+
<p>
−
mso-fareast-font-family:"Times New Roman";color:black;mso-themecolor:text1;
+
−
mso-ansi-language:EN-US'>9/10</span><span lang=EN-US style='font-family:Times;
+
</p>
−
mso-bidi-font-family:"Times New Roman";color:black;mso-themecolor:text1;
+
<p>
−
mso-ansi-language:EN-US'><o:p></o:p></span></p>
+
 +
</p>
 +
<ol>
  
−
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
+
<li>Transformed gib 4.1 gib 4.2
−
mso-fareast-font-family:"Times New Roman";color:black;mso-themecolor:text1;
+
</li>
−
mso-ansi-language:EN-US'>Filtered Vitamin B12<o:p></o:p></span></p>
+
</ol>
 +
<p>
 +
Overnight ligation sgrna McyI petblue
 +
</p>
 +
<p>
 +
Sgrna Mcyb PETblue
 +
</p>
 +
<p>
 +
gib -0.9ul dna
 +
</p>
 +
<p>
 +
    sgrna -2ul
 +
</p>
 +
<p>
 +
 +
</p>
 +
<ol>
  
−
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
+
<li>Checked plates for previous transformation: gib 3 in 1 -  
−
mso-fareast-font-family:"Times New Roman";color:black;mso-themecolor:text1;
+
</li>
−
mso-ansi-language:EN-US'>Autoclave bold3n<o:p></o:p></span></p>
+
</ol>
 +
<p>
 +
  gib 2    - no colonies
 +
</p>
 +
<p>
 +
  gib 3    - from last year -PETblue stock -yes colonies
 +
</p>
 +
<p>
 +
 +
</p>
 +
<ol>
  
−
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
+
<li>Picked 6 colonies from petblue
−
mso-fareast-font-family:"Times New Roman";color:black;mso-themecolor:text1;
+
</li>
−
mso-ansi-language:EN-US'>Add to agar</span><span lang=EN-US style='font-size:
+
</ol>
−
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>
+
petblue 1-3 -3ml(with amp)
 +
</p>
 +
<p>
 +
petblue 4-5 -2ml       
 +
</p>
 +
<p>
 +
Master plate -no xgal iptg
 +
</p>
 +
<p>
 +
 +
</p>
 +
<ol>
  
−
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
+
<li>Run gel for PCR products 
−
mso-fareast-font-family:"Times New Roman";color:black;mso-themecolor:text1;
+
</li>
−
mso-ansi-language:EN-US'><o:p>&nbsp;</o:p></span></p>
+
</ol>
 +
<p>
 +
Fragment 2, Fragment 3 x gib
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
Gel purification
 +
</p>
 +
<p>
 +
Dna concentration on 13/8
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
15 August 
 +
</p>
 +
<p>
 +
 +
</p>
 +
<ol>
  
−
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
+
<li>Restriction endonuclease cut for Fragment1 Fragment 2 and Fragment 3
−
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
+
</li>
−
EN-US'>10/10<o:p></o:p></span></p>
+
</ol>
 +
<p>
 +
Xgib ~6 hr (MluI, BssaI and NheI)
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
nanodrop before restriction endonuclease cut
 +
</p>
 +
<p>
 +
 +
</p>
 +
<ol>
  
−
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
+
<li>Set up gibson reaction
−
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
+
−
EN-US'>Plasmid purification with ethanol <span class=SpellE>percipitation</span><o:p></o:p></span></p>
+
  
−
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
+
<li>Fragment 3 50ng      16ul-  
−
"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:
+
<li>Fragment 2 38.73ng 2.1 ul-  
−
"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:
+
<li>Fragment 1 29.26ng 0.4ul-20ul/xh
−
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
+
−
EN-US'>11/10<o:p></o:p></span></p>
+
  
−
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
+
<li>Nebuilder                10ul-  
−
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
+
−
EN-US'>Measure Optical density (growth curve)<o:p></o:p></span></p>
+
  
−
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
+
<li>H2o                      5.9ul-  
−
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
+
</li>
−
EN-US'>Transformation of Microcystis<o:p></o:p></span></p>
+
</ol>
 +
<p>
 +
 +
</p>
 +
<p>
 +
50c for 2hr (optimized protocol)
 +
</p>
 +
<p>
 +
 +
</p>
 +
<ol>
  
−
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
+
<li>Plasmid purification of petblue 
−
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
+
</li>
−
EN-US'>Electroporation<o:p></o:p></span></p>
+
</ol>
 +
<p>
 +
            elute using 30ul 
 +
</p>
 +
<p>
 +
nanodrop
 +
</p>
 +
<p>
 +
 +
</p>
 +
<ol>
  
−
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
+
<li>Gib 4.1 -petblue mcyb plasmid have 1 colony each
−
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
+
</li>
−
EN-US'>Ligation by Peter<o:p></o:p></span></p>
+
</ol>
 +
<p>
 +
-picked & inoculated
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
 +
</p>
 +
<ol>
  
−
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
+
<li>PCR purified restriction endonuclease cuts from (Fragment 1, 2, 3 13/8?)
−
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
+
</li>
−
EN-US'>DNA purification <o:p></o:p></span></p>
+
</ol>
 +
<p>
 +
-ligated h3 & 3 in 1
 +
</p>
 +
<p>
 +
1+3 1:1
 +
</p>
 +
<p>
 +
1+2+31:1:1
 +
</p>
 +
<p>
 +
 +
</p>
 +
<ol>
  
−
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
+
<li>Sequencing
−
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
+
</li>
−
EN-US'><o:p>&nbsp;</o:p></span></p>
+
</ol>
 +
<p>
 +
 +
</p>
 +
<p>
 +
16 August
 +
</p>
 +
<p>
 +
 +
</p>
 +
<ol>
  
−
<p class=MsoNormal style='tab-stops:283.0pt'><span lang=EN-US style='font-family:
+
<li>Plasmid purification of 
−
Times;mso-bidi-font-family:"Times New Roman";mso-ansi-language:EN-US'>14/10</span><span
+
</li>
−
lang=EN-US style='font-family:"Times New Roman",serif;mso-fareast-font-family:
+
</ol>
−
"Times New Roman";mso-ansi-language:EN-US'><o:p></o:p></span></p>
+
<p>
 +
Gib4.1 & pet blue mcyb plasmid from 1518
 +
</p>
 +
<p>
 +
Gib4.1 does not seem to grow
 +
</p>
 +
<p>
 +
During inoculation
 +
</p>
 +
<p>
 +
Therefore, low yield.
 +
</p>
 +
<p>
 +
 +
</p>
 +
<ol>
  
−
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
+
<li>Run gel for Fragment 1+3 & 3in1 (ligated)
−
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Transformation
+
</li>
−
of previous ligation<o:p></o:p></span></p>
+
</ol>
 +
<p>
 +
 +
</p>
 +
<ol>
  
−
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
+
<li>Intended to amp 1+3 using 3f + 1r and 3r + 1f
−
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'><o:p>&nbsp;</o:p></span></p>
+
</li>
 +
</ol>
 +
<p>
 +
 +
</p>
 +
<ol>
  
−
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
+
<li>Intended to check gib4 using 1+2, 2+3, 1+3 primers.
−
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>15/10<o:p></o:p></span></p>
+
</li>
 +
</ol>
 +
<p>
 +
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
(3) and (4) failed possibily due to PCR errors
 +
</p>
 +
<p>
 +
-use 3 mins extension 65c other times and maximum
 +
</p>
 +
<p>
 +
 +
</p>
 +
<ol>
  
−
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
+
<li>Intended to check sgrna mcyb petblue using colony PCR
−
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Pick clones,
+
</li>
−
inoculate i12<o:p></o:p></span></p>
+
</ol>
 +
<p>
 +
(failed due to 0.7% gel, not 1.5% gel pic in chelsy’s phone
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
19 August 
 +
</p>
 +
<p>
 +
 +
</p>
 +
<ol>
  
−
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
+
<li>Colony PCR for mcyb sgrna petblue
−
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Standard
+
</li>
−
solution<o:p></o:p></span></p>
+
</ol>
 +
<p>
 +
(only 1 white colony)
 +
</p>
 +
<p>
 +
Results positive: band size ~300bp
 +
</p>
 +
<p>
 +
 +
</p>
 +
<ol>
  
−
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
+
<li>Inoculated 2 3ml tubes of gib4
−
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'><o:p>&nbsp;</o:p></span></p>
+
</li>
 +
</ol>
 +
<p>
 +
-gib4.1 & 4.2
 +
</p>
 +
<p>
 +
From master plate
 +
</p>
 +
<p>
 +
 +
</p>
 +
<ol>
  
−
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
+
<li>Set up overnight PCR to amp mcyi, Fragment1, Fragment3 xgib,  
−
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>16/10<o:p></o:p></span></p>
+
</li>
 +
</ol>
 +
<p>
 +
 +
</p>
 +
<ol>
  
−
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
+
<li>Restriction endonuclease (PstI, HindIII, MluI, BssaI, NheI) cut mcyi (247.8ng), petblue (1000ng), Fragment1 (741ng)- Fragment3 (140ng) overnight [mcyi only added 3ul]
−
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Pour plate
+
</li>
−
with kanamycin resistance and chloramphenicol<span style='mso-spacerun:yes'>  
+
</ol>
−
</span><o:p></o:p></span></p>
+
<p>
 +
 +
</p>
 +
<ol>
  
−
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
+
<li>Checked neb 3 in 1 using different primers 
−
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Takara
+
</li>
−
plasmid purification of insert 1 and insert 2<o:p></o:p></span></p>
+
</ol>
 +
<p>
 +
Checked Fragment 1+3
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
20 August 
 +
</p>
 +
<p>
 +
 +
</p>
 +
<ol>
  
−
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
+
<li>Run gel for mcyi, Fragment1-3 xgib
−
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'><o:p>&nbsp;</o:p></span></p>
+
</li>
 +
</ol>
 +
<p>
 +
-gel purification takara
 +
</p>
 +
<p>
 +
 +
</p>
 +
<ol>
  
−
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
+
<li>PCR clean mcyi, petblue 2 from overnight restriction endonuclease cut
−
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>17/10<o:p></o:p></span></p>
+
</li>
 +
</ol>
 +
<p>
 +
 +
</p>
 +
<ol>
  
−
<p class=MsoNormal style='line-height:107%'><span lang=EN-US style='font-family:
+
<li>Ligated mcyi petblue, Fragment1+3
−
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
+
</li>
−
EN-US'>Peter ethanol precipitation of final product<o:p></o:p></span></p>
+
</ol>
 +
<p>
 +
-vector 100ng-vector 100ng
 +
</p>
 +
<p>
 +
1:3 1:1
 +
</p>
 +
<p>
 +
 +
</p>
 +
<ol>
  
−
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
+
<li>?
−
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Electroporation
+
</li>
−
of Microcystis Aeruginosa UTEX 2388<o:p></o:p></span></p>
+
</ol>
 +
<p>
 +
 +
</p>
 +
<ol>
  
−
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
+
<li>Plasmid purification gib6.4
−
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Ligation
+
</li>
−
mixture (cut with <span class=SpellE>SpelI</span>, <span class=SpellE>xbaI</span>)<o:p></o:p></span></p>
+
</ol>
 +
<p>
 +
-yield extremely low
 +
</p>
 +
<p>
 +
 +
</p>
 +
<ol>
  
−
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
+
<li>Picked colonies: 2 nebuilder 3in1.-
−
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Run gel for
+
</li>
−
plasmid purification product of I2<o:p></o:p></span></p>
+
</ol>
 +
<p>
 +
    2 gib4  -master plate
 +
</p>
 +
<p>
 +
  2 sgrna mcyb petblue-  
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
21 August
 +
</p>
 +
<p>
 +
 +
</p>
 +
<ol>
  
−
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
+
<li>Plasmid pur for 6 tubes -nebuilder x2, gib4 x2, sgrna mcyb petblue
−
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Nanodrop I2<o:p></o:p></span></p>
+
</li>
 +
</ol>
 +
<p>
 +
 +
</p>
 +
<ol>
  
−
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
+
<li>Collected overnight ligation
−
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Anson adjust
+
</li>
−
pH for BG11<o:p></o:p></span></p>
+
</ol>
 +
<p>
 +
-amplified Fragment 1+3 wing
 +
</p>
 +
<p>
 +
Primer Fragment1 f & primer Fragment3 r (vice versa also)
 +
</p>
 +
<p>
 +
 +
</p>
 +
<ol>
  
−
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
+
<li>Transformed sgrna mcyi petblue and dcas9 gfp (l3)
−
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Error: labelling
+
</li>
−
error for plasmid purification<o:p></o:p></span></p>
+
</ol>
 +
<p>
 +
 +
</p>
 +
<ol>
  
−
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
+
<li>Received sanger sequence results 
−
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'><o:p>&nbsp;</o:p></span></p>
+
</li>
 +
</ol>
 +
<p>
 +
-99.4% identical
 +
</p>
 +
<p>
 +
mutation- x
 +
</p>
 +
<p>
 +
 +
</p>
 +
<ol>
  
−
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
+
<li>Picked 25 colonies from previous l3 plate
−
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>18/10<o:p></o:p></span></p>
+
</li>
 +
</ol>
 +
<p>
 +
-colony PCR 25-49  -one taq
 +
</p>
 +
<p>
 +
50- sapphire
 +
</p>
 +
<p>
 +
 +
</p>
 +
<ol>
  
−
<p class=MsoNormal><span lang=EN-US style='font-family:"Times New Roman",serif;
+
<li>Single cut & double cut 3 in 1 plasmid
−
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'>Pick insert
+
</li>
−
1 and <span class=GramE>2 ,BL</span>21 and NEB5</span><span lang=EN-US
+
</ol>
−
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:
+
-all undesired results
−
"Times New Roman",serif;mso-fareast-font-family:"Times New Roman";mso-ansi-language:
+
</p>
−
EN-US'> clones <o:p></o:p></span></p>
+
<p>
 +
 +
</p>
 +
<p>
 +
22 Aug
 +
</p>
 +
<p>
 +
gibson assembly ,incubate 2h
 +
</p>
 +
<p>
 +
run gel for colony PCR and Fragment 1+3 and the overnight cut of 3 in 1
 +
</p>
 +
<p>
 +
For colonies with correct band after colony PCR, pick colony from master plate, inoculate
 +
</p>
 +
<p>
 +
Fragment 1+3, Fragment 2 re cut,
 +
</p>
 +
<p>
 +
Send mcyb to sequencing
 +
</p>
 +
<p>
 +
Pick colony from plates  if have
 +
</p>
 +
<p>
 +
Transform gibson products
 +
</p>
 +
<p>
 +
Ligate Fragment 2 to 1+3
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
23 Aug
 +
</p>
 +
<p>
 +
Run gel for PCR dcas9 gfp, 
 +
</p>
 +
<p>
 +
Run gel for PCR 1+3 sgrna mcyi pet blue
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
26 Aug
 +
</p>
 +
<p>
 +
Run gel for previous 40 colony PCR
 +
</p>
 +
<p>
 +
Inoculate l3 with correct band size
 +
</p>
 +
<p>
 +
Preparation for electroporation
 +
</p>
 +
<p>
 +
Transform ligation product and 3 in 1 for characterization
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
27 Aug
 +
</p>
 +
<p>
 +
Pick clone for previous transformation
 +
</p>
 +
<p>
 +
3 in 1 plasmid pur using traditional method
 +
</p>
 +
<p>
 +
Restriction endonuclease cut gib 5 (MluI)
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
28 Aug
 +
</p>
 +
<p>
 +
PCR screening mcyi, b
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
29 Aug
 +
</p>
 +
<p>
 +
Plasmid purification. Using tradition n takara
 +
</p>
 +
<p>
 +
Inoculate gib 5, prepare for measurement
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
30 Aug 
 +
</p>
 +
<p>
 +
I think no lab
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
3/9
 +
</p>
 +
<p>
 +
Restriction endonuclease cut dcas9 gfp (EcoRI, PstI)
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
4/9
 +
</p>
 +
<p>
 +
Run gel for restriction endonuclease cut dcas9 gfp
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
5/9
 +
</p>
 +
<p>
 +
Transformation inoculate pet blue, puc19, 3 in 1, psb1c3 transformants
 +
</p>
 +
<p>
 +
Plasmid purification 3 in 1 using takara plasmid purification kit
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
6/9
 +
</p>
 +
<p>
 +
Plasmid purification 3 in 1 using takara plasmid purification kit
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
9/9
 +
</p>
 +
<p>
 +
2 restriction endonuclease cuts (dCas9-GFP-sgRNA)
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
10/9
 +
</p>
 +
<p>
 +
Run gel for the colony PCR products (1.5 gel, all in) 65v 45 min 700 or no band
 +
</p>
 +
<p>
 +
Pick colony and colony PCR 
 +
</p>
 +
<p>
 +
Pour agar plate 
 +
</p>
 +
<p>
 +
take the 2 restriction endonuclease cuts from 37c n run gel (1.5 gel 65v 45 min) shd be ~5500bp and ~3500bp
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
11/9
 +
</p>
 +
<p>
 +
PCR clean for 3 in 1 cut
 +
</p>
 +
<p>
 +
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)
 +
</p>
 +
<p>
 +
Gel purification for the above cut gel (gm buffer 650ul)
 +
</p>
 +
<p>
 +
Run gel for colony PCR yesterday (1.5 gel)
 +
</p>
 +
<p>
 +
Set ligation mixture (restriction endonuclease cuts of  3 in 1 and mcy dcas9 gfp) 1:1
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
12/9
 +
</p>
 +
<p>
 +
Heat inactivate ligation
 +
</p>
 +
<p>
 +
2. Transform ligation (total dna 60ng)
 +
</p>
 +
<p>
 +
3. Dilute overnight inoculation, 1 to 3
 +
</p>
 +
<p>
 +
4. Set restriction endonuclease cut of 3 in 1 and sgrna dcas9 gfp
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
13/9
 +
</p>
 +
<p>
 +
Measure od (growth curve) for modelling
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
16/9
 +
</p>
 +
<p>
 +
Restriction endonuclease cuts for final product (XhoI, XbaI)
 +
</p>
 +
<p>
 +
Pick colony for growth curve
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
17/9
 +
</p>
 +
<p>
 +
Growth curve
 +
</p>
 +
<p>
 +
Run gel for the restriction endonuclease cuts, PCR clean, start ligation
 +
</p>
 +
<p>
 +
Transform ligation mixture
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
18/9
 +
</p>
 +
<p>
 +
Transofrmation of final ligation products
 +
</p>
 +
<p>
 +
2. Od measurements
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
19/9
 +
</p>
 +
<p>
 +
pick colony (fat clone)
 +
</p>
 +
<p>
 +
Transformation final product
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
20/9
 +
</p>
 +
<p>
 +
Pick clone
 +
</p>
 +
<p>
 +
PCR check band 
 +
</p>
 +
<ul>
  
−
<p class=MsoListParagraph><span lang=EN-US style='font-family:"Times New Roman",serif;
+
<li>Correct 
−
mso-fareast-font-family:"Times New Roman";mso-ansi-language:EN-US'><o:p>&nbsp;</o:p></span></p>
+
</li>
 +
</ul>
 +
<p>
 +
Plasmid purification by PETER failed cuz me flip his gel…
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
23/9
 +
</p>
 +
<p>
 +
Pick colony of final product
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
24/9
 +
</p>
 +
<p>
 +
Plasmid purification
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
25/9 
 +
</p>
 +
<p>
 +
Nanodrop final product
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
26/9
 +
</p>
 +
<p>
 +
Restriction endonuclease cut for final product (PstI)
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
27/9
 +
</p>
 +
<p>
 +
Run gel for the restriction endonuclease cuts and final product 24 and 15. 
 +
</p>
 +
<p>
 +
All in for restriction endonuclease cuts,
 +
</p>
 +
<p>
 +
5ul for final product 24 and 15
 +
</p>
 +
<p>
 +
65v 50min
 +
</p>
 +
<p>
 +
Nanodrop final product
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
30/9
 +
</p>
 +
<p>
 +
Transformation
 +
</p>
 +
<p>
 +
final product plasmid 15 17 24 29(bl21)
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
3/10
 +
</p>
 +
<p>
 +
Observe green fluorescence by uv exposure
 +
</p>
 +
<p>
 +
Inoculate
 +
</p>
 +
<p>
 +
Observe green fluorescence again by uv exposure
 +
</p>
 +
<ul>
  
 +
<li>All negative 
 +
</li>
 +
</ul>
 +
<p>
 +
 +
</p>
 +
<p>
 +
4/10
 +
</p>
 +
<p>
 +
Plasmid purification of final product
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
7/10
 +
</p>
 +
<p>
 +
Transformation of final products into DH5a
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
8/10
 +
</p>
 +
<p>
 +
Prepared 0.1mm nacl, 0.1mm hepes, 0.1mm vitb12, all not sterilized. Picked 10 colonies (final product) , inoculated in 20ml LB 
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
9/10
 +
</p>
 +
<p>
 +
Filtered Vitamin B12
 +
</p>
 +
<p>
 +
Autoclave bold3n
 +
</p>
 +
<p>
 +
Add to agar
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
 
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
10/10
 +
</p>
 +
<p>
 +
Plasmid purification with ethanol
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
11/10
 +
</p>
 +
<p>
 +
Measure Optical density (growth curve)
 +
</p>
 +
<p>
 +
Transformation of microcystis
 +
</p>
 +
<p>
 +
Electroporation
 +
</p>
 +
<p>
 +
Ligation by Peter
 +
</p>
 +
<p>
 +
Dna purification 
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
14/10
 +
</p>
 +
<p>
 +
Transformation of previous ligation
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
15/10
 +
</p>
 +
<p>
 +
Pick clones, inoculate i12
 +
</p>
 +
<p>
 +
Standard solution
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
16/10
 +
</p>
 +
<p>
 +
Pour plate with kanamycin resistance and chloramphenicol 
 +
</p>
 +
<p>
 +
Takara plasmid purification of insert 1 and insert 2
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
17/10
 +
</p>
 +
<p>
 +
Peter ethanol precipitation of final product
 +
</p>
 +
<p>
 +
Electroporation (natural tranformation protocol)
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
Ligation mixture (cut with SpelI, xbaI)
 +
</p>
 +
<p>
 +
Run gel for plasmid purifiction product of i12
 +
</p>
 +
<p>
 +
Nanodrop i12
 +
</p>
 +
<p>
 +
Anson adjust pH for bg11
 +
</p>
 +
<p>
 +
Error: labelling error for plasmid pu
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
18/10
 +
</p>
 +
<p>
 +
Pick insert 1 and 2 ,BL21 and NEB5a clones 
 +
</p>
 +
<p>
 +
 +
</p>
 
</div>
 
</div>
  

Latest revision as of 03:25, 22 October 2019

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JUDGING FORM ⇗

17 July

Transformation of dcas9 plasmid 2019 kit plate 5 well 22n.

- kanamycin resistance plate

- to E. coli NEB5a

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

  1. Assembling Fragment 1+2+3

-restriction endonuclease cut + ligation

NheI and BssSI-v2

  1. Dcas9 preparation clean
  2. Run gel NEB5α

Transformed produce to E.coli neb5

  1. Cloned GFP to dcas9

-restriction endonuclease cut + ligation

EcorI NsiI

Transformed product to E.coli neb5

  1. 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

- gfp+ dcas9 plasmid again as l3

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

  1. Checked plates for sgrna pet blue mcyi

Sgrna pet blue mcyI2

Sgrna pet blue mcyb 2

3 in 1

Gib 2.1

Gib 2.2

-all no colony.

  1. Conducted PCR purification of overnight (14h) restriction endonuclease cut of dcas9 gfp (EcoRI, NsiI)

-50 rxn mixture

  1. Conducted ligation

1:3 & 1:5

Vector 250ng

Both 20ul rxn,

-rm temp for 10 min

heat inactive at 65c for 10 min

on ice 90min

-then add ligase again

ligate over weekend.

  1. Amplified Fragment1 + 2 + 3

-sgrna I, sgrna B, gfp

For gibson

Fragment 1 2 3 gfp, 65c

Sgrnas 55c

All 50 ul rxn

  1. Weight powder, for soc (800ml no glucose/ mgxx) (no yellow)

-LB agar (have yellow) 1800ul

Glucose 9g

  1. Run gel for all 6 Fragments (all in)

-75v 45 mins

Ladder use 1kb & 50bp

Loading dye 6x

  1. Gel purification -elude using 30ul elution b.

12 August

  1. Colony PCR for those without bands reaction

-picked colonies from master plate

  1. Performed gibson(gib3) & nebuilder
  2. Scaled down to 10ml rxn
  3. Concentration acc to following nanodrop

-incubation 1h

  1. PCR for xgib 1 (Fragment1) gib1 (Fragment1)

xgib 2 (Fragment2) gib2 (Fragment2)

xgib 3 (Fragment3) gib3 (Fragment3)

  1. Colony PCR no.24 correct band

(dcas9 gfp) L3

band size 700-800 bp

inoculated 24 Hours (picked from master plate)

  1. 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

  1. Run gel for previous PCR

  1. Checked plates from 12/8

Colony: petblue +ve

dCas9 gfp 1:3

dCas9 gfp 1:5

  1. Plasmid purification l3

dCas9-GFP 24

Re cut for Fragment1-3 (gibson assembly)

McyI, Mcyb

14 August

  1. Run gel for xgib Fragment 1

-no bands at all

Possibly due to pipetting errors.

  1. 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

98c 98c 66c 72c 72c 4c
30sec 5s 15s 1.5min 2min

  1. Transformed gib 4.1 gib 4.2

Overnight ligation sgrna McyI petblue

Sgrna Mcyb PETblue

gib -0.9ul dna

sgrna -2ul

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

gib 2 - no colonies

gib 3 - from last year -PETblue stock -yes colonies

  1. Picked 6 colonies from petblue

petblue 1-3 -3ml(with amp)

petblue 4-5 -2ml

Master plate -no xgal iptg

  1. Run gel for PCR products

Fragment 2, Fragment 3 x gib

Gel purification

Dna concentration on 13/8

15 August

  1. Restriction endonuclease cut for Fragment1 Fragment 2 and Fragment 3

Xgib ~6 hr (MluI, BssaI and NheI)

nanodrop before restriction endonuclease cut

  1. Set up gibson reaction
  2. Fragment 3 50ng 16ul-
  3. Fragment 2 38.73ng 2.1 ul-
  4. Fragment 1 29.26ng 0.4ul-20ul/xh
  5. Nebuilder 10ul-
  6. H2o 5.9ul-

50c for 2hr (optimized protocol)

  1. Plasmid purification of petblue

elute using 30ul

nanodrop

  1. Gib 4.1 -petblue mcyb plasmid have 1 colony each

-picked & inoculated

  1. PCR purified restriction endonuclease cuts from (Fragment 1, 2, 3 13/8?)

-ligated h3 & 3 in 1

1+3 1:1

1+2+31:1:1

  1. Sequencing

16 August

  1. Plasmid purification of

Gib4.1 & pet blue mcyb plasmid from 1518

Gib4.1 does not seem to grow

During inoculation

Therefore, low yield.

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

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

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

(3) and (4) failed possibily due to PCR errors

-use 3 mins extension 65c other times and maximum

  1. Intended to check sgrna mcyb petblue using colony PCR

(failed due to 0.7% gel, not 1.5% gel pic in chelsy’s phone

19 August

  1. Colony PCR for mcyb sgrna petblue

(only 1 white colony)

Results positive: band size ~300bp

  1. Inoculated 2 3ml tubes of gib4

-gib4.1 & 4.2

From master plate

  1. Set up overnight PCR to amp mcyi, Fragment1, Fragment3 xgib,

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

  1. Checked neb 3 in 1 using different primers

Checked Fragment 1+3

20 August

  1. Run gel for mcyi, Fragment1-3 xgib

-gel purification takara

  1. PCR clean mcyi, petblue 2 from overnight restriction endonuclease cut

  1. Ligated mcyi petblue, Fragment1+3

-vector 100ng-vector 100ng

1:3 1:1

  1. ?

  1. Plasmid purification gib6.4

-yield extremely low

  1. Picked colonies: 2 nebuilder 3in1.-

2 gib4 -master plate

2 sgrna mcyb petblue-

21 August

  1. Plasmid pur for 6 tubes -nebuilder x2, gib4 x2, sgrna mcyb petblue

  1. Collected overnight ligation

-amplified Fragment 1+3 wing

Primer Fragment1 f & primer Fragment3 r (vice versa also)

  1. Transformed sgrna mcyi petblue and dcas9 gfp (l3)

  1. Received sanger sequence results

-99.4% identical

mutation- x

  1. Picked 25 colonies from previous l3 plate

-colony PCR 25-49 -one taq

50- sapphire

  1. 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

Pick colony from plates if have

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 pet blue

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 pur using traditional method

Restriction endonuclease cut gib 5 (MluI)

28 Aug

PCR screening mcyi, b

29 Aug

Plasmid purification. Using tradition n takara

Inoculate gib 5, prepare for measurement

30 Aug

I think no lab

3/9

Restriction endonuclease cut dcas9 gfp (EcoRI, PstI)

4/9

Run gel for restriction endonuclease cut dcas9 gfp

5/9

Transformation inoculate pet blue, 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

2. Od measurements

19/9

pick colony (fat clone)

Transformation final product

20/9

Pick clone

PCR check band

  • Correct

Plasmid purification by PETER failed cuz me flip his gel…

23/9

Pick colony of final product

24/9

Plasmid purification

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

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 (natural tranformation protocol)

Ligation mixture (cut with SpelI, xbaI)

Run gel for plasmid purifiction product of i12

Nanodrop i12

Anson adjust pH for bg11

Error: labelling error for plasmid pu

18/10

Pick insert 1 and 2 ,BL21 and NEB5a clones