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

(Prototype team page)
 
Line 4: Line 4:
  
 
<div class="column full_size">
 
<div class="column full_size">
 +
<p>
 +
<strong>17 July                                                                    </strong>
 +
</p>
 +
<p>
 +
<strong>Transformation of dCas9 plasmid 2019 kit plate 5 well 22N. </strong>
 +
</p>
 +
<p>
 +
<strong>- Kanamycin Resistance plate</strong>
 +
</p>
 +
<p>
 +
<strong>- to E. Coli Neb 5a  </strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong>19 July</strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong>Picked 6 colonies</strong>
 +
</p>
 +
<p>
 +
<strong>-Grow overnight in LB</strong>
 +
</p>
 +
<p>
 +
<strong>Plasmid Purification (Takara)</strong>
 +
</p>
 +
<p>
 +
<strong>-Colony 1 & 2 did not grow</strong>
 +
</p>
 +
<p>
 +
<strong>-Run agarose gel 100V 30 mins</strong>
 +
</p>
 +
<p>
 +
<strong>-Eluted DNA using 30uL elution buffer</strong>
 +
</p>
 +
<p>
 +
<strong>  -Nanodrop      </strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong>22 July</strong>
 +
</p>
 +
<p>
 +
<strong>Plasmid prep</strong>
 +
</p>
 +
<p>
 +
<strong>Run gel for dcas9</strong>
 +
</p>
 +
<p>
 +
<strong>Resuspend dna</strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong>23 July</strong>
 +
</p>
 +
<p>
  
−
<h1>Notebook</h1>
+
        <strong>1) Assembling frag1 +2 +3</strong>
−
<p> Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.</p>
+
</p>
 +
<p>
  
−
</div>
+
    <strong>-RE cut + ligation</strong>
−
<div class="clear"></div>
+
</p>
 +
<p>
  
 +
    <strong>NheI MliI BssSI-v2</strong>
 +
</p>
 +
<p>
  
 +
    <strong> </strong>
 +
</p>
 +
<p>
  
−
<div class="column two_thirds_size">
+
        <strong>2) dcas9 preparation Clean</strong>
−
<h3>What should this page have?</h3>
+
</p>
−
<ul>
+
<p>
−
<li>Chronological notes of what your team is doing.</li>
+
−
<li> Brief descriptions of daily important events.</li>
+
−
<li>Pictures of your progress. </li>
+
−
<li>Mention who participated in what task.</li>
+
−
</ul>
+
  
−
</div>
+
        <strong>3) Run gel Anson 5α</strong>
 +
</p>
 +
<p>
  
−
<div class="column third_size">
+
    <strong> </strong>
−
<div class="highlight decoration_A_full">
+
</p>
−
<h3>Inspiration</h3>
+
<p>
−
<p>You can see what others teams have done to organize their notes:</p>
+
  
−
<ul>  
+
    <strong> </strong>
−
<li><a href="https://2018.igem.org/Team:Munich/Notebook">2018 Munich</a></li>
+
</p>
−
<li><a href="https://2014.igem.org/Team:ATOMS-Turkiye/Notebook">2014 ATOMS-Turkiye</a></li>
+
<p>
−
<li><a href="https://2014.igem.org/Team:Tec-Monterrey/ITESM14_project.html#tab_notebook">2014 Tec Monterrey</a></li>
+
<strong>Transformed produce to Ecoli NEB5a</strong>
−
<li><a href="https://2014.igem.org/Team:Kyoto/Notebook/Magnetosome_Formation#title">2014 Kyoto</a></li>
+
</p>
−
<li><a href="https://2014.igem.org/Team:Cornell/notebook">2014 Cornell</a></li>
+
<p>
−
</ul>
+
<strong> </strong>
−
</div>
+
</p>
−
</div>
+
<p>
 +
 
 +
        <strong>4) Cloned GFP to dCas9</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong>-RE cut + ligation</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong>EcoRI NsiI</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong> </strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong>Transformed product to Ecoli NEB5a</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong> </strong>
 +
</p>
 +
<p>
 +
 
 +
        <strong>5) Transformed PUC19 from stock (for cloning sgRNA later)</strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong>1 Aug</strong>
 +
</p>
 +
<p>
 +
<strong>Cyanobac arrive, cultured in 15x4 mL medium</strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong>2 Aug</strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong>6 Aug</strong>
 +
</p>
 +
<p>
 +
<strong>Colony PCR</strong>
 +
</p>
 +
<p>
 +
<strong>PCR Clean, RE ligation</strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong>7 Aug</strong>
 +
</p>
 +
<p>
 +
<strong>-gel purification after 75V 45min 0.7 gel</strong>
 +
</p>
 +
<p>
 +
<strong>- Gibson assembly</strong>
 +
</p>
 +
<p>
 +
<strong>- Run Gel for Overnigt SgRNA PCR product</strong>
 +
</p>
 +
<p>
 +
<strong>- RE(2hrs incubation)+ligation for SgRNA and pETBlue</strong>
 +
</p>
 +
<p>
 +
<strong>-Transformed 3in1, pETBlue sgRNA and L3 into NEB5a</strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong>8 Aug</strong>
 +
</p>
 +
<p>
 +
<strong>Colony PCR, run gel, pick clone</strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong>9 August</strong>
 +
</p>
 +
<p>
 +
 
 +
        <strong>1) Checked plates for sgRNA pet blue mcyI</strong>
 +
</p>
 +
<p>
 +
 
 +
        <strong>sgRNA pet blue mcyI 2</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong>sgRNA pet blue mcyB 2</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong>3 in 1</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong>Gib 2.1</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong>Gib 2.2</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong>-All no colony.</strong>
 +
</p>
 +
<p>
 +
 
 +
        <strong>2) Conducted PCR purification of overnight (14h) RE cut of dCas9 GFP</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong>-50rxn mixture</strong>
 +
</p>
 +
<p>
 +
 
 +
        <strong>3) Conducted ligation</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong>1:3 & 1:5</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong>Vector 250ng</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong>Both 20uL rxn,</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong> </strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong>-rm temp for 10 min</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong>        Heat inactive at 65°C for 10 min</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong>        On ice 90min</strong>
 +
</p>
 +
<p>
 +
<strong>-Then add ligase again</strong>
 +
</p>
 +
<p>
 +
<strong>        Ligate over weekend.</strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
 
 +
        <strong>4) Amplified frag1 + 2 + 3</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong>-sgRNA I, sgRNA B, GFP</strong>
 +
</p>
 +
<p>
 +
 
 +
        <strong>For Gibson</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong>Frag 1 2 3 gfp, 65°C</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong>sgRNAs 55C</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong> </strong>
 +
</p>
 +
<p>
 +
 
 +
        <strong>All 50 uL rxn</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong> </strong>
 +
</p>
 +
<p>
 +
 
 +
        <strong>5) Weight powder, for SOC (800mL no glucose/ mgXX) (no yellow)</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong>-LB agar (have yellow) 1800uL</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong>Glucose 9g</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong> </strong>
 +
</p>
 +
<p>
 +
 
 +
        <strong>6) Run gel for all 6 frags (all in)</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong>-75V 45 mins</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong>Ladder use 1kb & 50bp</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong>Loading dye 6X</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong> </strong>
 +
</p>
 +
<p>
 +
 
 +
        <strong>7) Gel Purification -elude using 30uL elution b.</strong>
 +
</p>
 +
<p>
 +
<strong>12 August</strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
 
 +
        <strong>1) Colony PCR for those without bands reaction</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong>-Picked colonies from master plate</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong> </strong>
 +
</p>
 +
<p>
 +
 
 +
        <strong>2) Performed Gibson(Gib3) & NEBuilder</strong>
 +
</p>
 +
<p>
 +
 
 +
        <strong>-      Scaled down to 10mL rxn</strong>
 +
</p>
 +
<p>
 +
 
 +
        <strong>-      Concentration acc to following nanodrop</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong>-incubation 1h</strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
 
 +
        <strong>3) PCR for XGib 1 (frag1) Gib1 (frag1)</strong>
 +
</p>
 +
<p>
 +
<strong>                    XGib 2 (frag2) Gib2 (frag2)</strong>
 +
</p>
 +
<p>
 +
<strong>                    XGib 3 (frag3) Gib3 (frag3)</strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
 
 +
        <strong>4) Colony PCR no.24 correct band</strong>
 +
</p>
 +
<p>
 +
 
 +
        <strong>(dcas9 gfp) L3</strong>
 +
</p>
 +
<p>
 +
<strong>        Band size 700-800 bp</strong>
 +
</p>
 +
<p>
 +
<strong>        Inoculated 24 (picked from master plate)</strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
 
 +
        <strong>5) Transformed Gibson 1</strong>
 +
</p>
 +
<p>
 +
<strong>                          Gibson 2</strong>
 +
</p>
 +
<p>
 +
<strong>        Gibson 3.2 NEBuilder (1)</strong>
 +
</p>
 +
<p>
 +
<strong>        +ve control  petblue NEBuilder (2)</strong>
 +
</p>
 +
<p>
 +
<strong>                          dCas9 gfp 1:3</strong>
 +
</p>
 +
<p>
 +
<strong>                          dCas9 gfp 1:5</strong>
 +
</p>
 +
<p>
 +
<strong>                          Gibson 3.1</strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong>13 August</strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
 
 +
        <strong>1) Run gel for previous PCR</strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
 
 +
        <strong>2) Checked plates from 12/8</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong>Colony: petblue +ve</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong>        dCas9 gfp 1:3</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong>        dCas9 gfp 1:5</strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
 
 +
        <strong>3) Plasmid purification L3</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong>dCas9 gfp 24</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong> </strong>
 +
</p>
 +
<p>
 +
<strong>Re Cut for frag1-3 (gib)</strong>
 +
</p>
 +
<p>
 +
<strong>McyI, McyB</strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong>14 August</strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
 
 +
        <strong>1) Run Gel for X gib Frag 1</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong>-No bands at all</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong>Possibly due to pipetting errors.</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong> </strong>
 +
</p>
 +
<p>
 +
 
 +
        <strong>2) Started 10uL reaction using Gibson concentration according to following gel. (pic Gib4)</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong> </strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong>Reaction: Frag3: 50ng</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong>            Frag2: 38.73ng</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong>            Frag1: 29.26ng</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong>        Nebuilder: 5uL</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong>            H2O</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong> </strong>
 +
</p>
 +
<p>
 +
 
 +
        <strong>3) Set up PCR reaction for XGib Frag2</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong>XGib Frag3</strong>
 +
</p>
 +
<p>
 +
 +
</p>
 +
 
 +
<table>
 +
  <tr>
 +
  <td>98°C
 +
  </td>
 +
  <td>98°C
 +
  </td>
 +
  <td>66°
 +
  </td>
 +
  <td>72°C
 +
  </td>
 +
  <td>72°C
 +
  </td>
 +
  <td>4°C
 +
  </td>
 +
  </tr>
 +
  <tr>
 +
  <td>30sec
 +
  </td>
 +
  <td>5s
 +
  </td>
 +
  <td>15s
 +
  </td>
 +
  <td>1.5min
 +
  </td>
 +
  <td>2min
 +
  </td>
 +
  <td>¥
 +
  </td>
 +
  </tr>
 +
</table>
 +
 
 +
 
 +
<p>
 +
 
 +
    <strong> </strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong> </strong>
 +
</p>
 +
<p>
 +
 
 +
        <strong>4) Transformed Gib 4.1 Gib 4.2</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong>Overnight ligation sgRNA mcyI Petblue</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong>sgRNA mcyB petblue</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong> Gib -0.9uL DNA </strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong> sgRNA -2uL</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong> </strong>
 +
</p>
 +
<p>
 +
 
 +
        <strong>5) Checked plates for previous transformation: Gib 3 in 1 -</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong>                                                                          Gib 2  - No colonies</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong>                                                                          Gib 3        -</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong>  From last yr -petblue stock -yes colonies</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong> </strong>
 +
</p>
 +
<p>
 +
 
 +
        <strong>6) Picked 6 colonies from petblue</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong>              Petblue 1-3 -3mL    (with</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong>              Petblue 4-5 -2mL  amp)</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong>Master plate -No xgal Iptg</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong> </strong>
 +
</p>
 +
<p>
 +
 
 +
        <strong>7) Run gel for pcr products</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong>Frag 2, frag 3 X Gib</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong> </strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong>Gel purification</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong>DNA concentration on 13/8</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong> </strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong> </strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong> </strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong>15 August</strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
 
 +
        <strong>1) RE cut for frag1 frag 2 and frag 3</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong>X Gib ~6 hr</strong>
 +
</p>
 +
<p>
 +
<strong>        </strong>
 +
</p>
 +
<p>
 +
<strong>        Nanodrop before RE cut</strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
 
 +
        <strong>2) Set up Gibson reaction</strong>
 +
</p>
 +
<p>
 +
 
 +
            <strong>a. Frag 3 50nL 16uL              -      </strong>
 +
</p>
 +
<p>
 +
 
 +
            <strong>b. Frag 2 38.73ng 2.1 uL        -</strong>
 +
</p>
 +
<p>
 +
 
 +
            <strong>c. Frag 1 29.26ng 0.4uL        -      20uL/xh</strong>
 +
</p>
 +
<p>
 +
 
 +
            <strong>d. Nebuilder          10uL          -</strong>
 +
</p>
 +
<p>
 +
 
 +
            <strong>e. H2O              5.9uL        -</strong>
 +
</p>
 +
<p>
 +
 
 +
        <strong> </strong>
 +
</p>
 +
<p>
 +
 
 +
        <strong>50C for 2hr</strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
 
 +
        <strong>3) Plasmid Purification of Petblue</strong>
 +
</p>
 +
<p>
 +
<strong>                    Elude using 30uL</strong>
 +
</p>
 +
<p>
 +
<strong>        Nanodrop</strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
 
 +
        <strong>4) Gib 4.1 -Petblue mcyB plasmid have 1 colony each</strong>
 +
</p>
 +
<p>
 +
<strong>                    -Picked & inoculated</strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
 
 +
        <strong>5) PCR purified RE cuts from (nolning?)</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong>-ligated H3 & 3 in 1</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong>        1+3  1:1</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong>        1+2+3 1:1:1</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong> </strong>
 +
</p>
 +
<p>
 +
 
 +
        <strong>6) Sequencing</strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong>16 August</strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
 
 +
        <strong>1) Plasmid purification of</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong>Gib4.1 & pET blue mcyB plasmid from 1518</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong>Gib4.1 does not seem to grow</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong>During inoculation</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong>Therefore, low yield.</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong> </strong>
 +
</p>
 +
<p>
 +
 
 +
        <strong>2) Run gel for frag 1+3 & 3in1 (ligated)</strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
 
 +
        <strong>3) Intended to amp 1+3 using 3F + 1R and 3R + 1F</strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
 
 +
        <strong>4) Intended to check Gib4 using 1+2, 2+3, 1+3 primers.</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong> </strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong> </strong>
 +
</p>
 +
<p>
 +
<strong>(3) and (4) failed possibily due to pcr errors</strong>
 +
</p>
 +
<p>
 +
<strong>-Use 3 mins extension        65C other times and maximum</strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
 
 +
        <strong>5) Intended to check sgRNA mcyB petblue using colony PCR</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong>(failed due to 0.7% gel, not 1.5% gel pic in chelsy’s phone</strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong>19 August</strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
 
 +
        <strong>1) Colony PCR for McyB sgRNA petblue</strong>
 +
</p>
 +
<p>
 +
 
 +
        <strong>(only 1 white colony)</strong>
 +
</p>
 +
<p>
 +
 
 +
        <strong>Results positive: band size ~300bp</strong>
 +
</p>
 +
<p>
 +
 
 +
        <strong> </strong>
 +
</p>
 +
<p>
 +
 
 +
        <strong>2) Inoculated 2 3mL tubes of Gib4</strong>
 +
</p>
 +
<p>
 +
 
 +
        <strong>-Gib4.1 & 4.2</strong>
 +
</p>
 +
<p>
 +
 
 +
        <strong>From master plate</strong>
 +
</p>
 +
<p>
 +
 
 +
        <strong> </strong>
 +
</p>
 +
<p>
 +
 
 +
        <strong>3) Set up overnight PCR to amp mcyI, frag1, frag3 xgib,</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong> </strong>
 +
</p>
 +
<p>
 +
 
 +
        <strong>4) RE cut mcyI (247.8ng), petblue (1000ng), frag1 (741ng)- frag3 (140ng) overnight [McyI only added 3uL]</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong> </strong>
 +
</p>
 +
<p>
 +
 
 +
        <strong>5) Checked Neb 3 in 1 using different primers</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong>Checked frag 1+3</strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong> 20 August</strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
 
 +
        <strong>1) Run gel for mcyI, frag1-3 xGib</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong>-gel purification Takara</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong> </strong>
 +
</p>
 +
<p>
 +
 
 +
        <strong>2) PCR clean mcyI, petblue 2 from overnight RE</strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
 
 +
        <strong>3) Ligated mcyI petblue, frag1+3</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong>-vector 100ng  -vector 100ng</strong>
 +
</p>
 +
<p>
 +
<strong>        1:3                      1:1</strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
 
 +
        <strong>4) ?</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong> </strong>
 +
</p>
 +
<p>
 +
 
 +
        <strong>5) Plasmid pur Gib6.4</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong>-yield extremely low</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong> </strong>
 +
</p>
 +
<p>
 +
 
 +
        <strong>6) Picked colonies: 2 Nebuilder 3in1.            -</strong>
 +
</p>
 +
<p>
 +
<strong>                                  2 Gib4                          -      Master Plate</strong>
 +
</p>
 +
<p>
 +
<strong>                                  2 sgRNA mcyB petblue  -</strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong>21 August</strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
 
 +
        <strong>1) Plasmid pur for 6 tubes -NEbuilder x2, gib4 x2, sgRNA mcyB petblue</strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
 
 +
        <strong>2) Collected overnight ligation</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong>-Amplified frag 1+3 wing</strong>
 +
</p>
 +
<p>
 +
<strong>Primer frag1 F & primer frag3 R (vice versa also)</strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
 
 +
        <strong>3) Transformed sgRNA mcyI petblue and dCas9 gfp (L3)</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong> </strong>
 +
</p>
 +
<p>
 +
 
 +
        <strong>4) Received sanger sequence results</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong>-99.4% identical</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong>        Mutation- X</strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
 
 +
        <strong>5) Picked 25 colonies from previous L3 plate</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong>-colony PCR          25-49  -one Taq</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong>                                50    - sapphire</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong> </strong>
 +
</p>
 +
<p>
 +
 
 +
        <strong>6) Single cut & double cut 3 in 1 plasmid</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong>-All undesired results</strong>
 +
</p>
 +
<p>
 +
 
 +
    <strong> </strong>
 +
</p>
 +
<p>
 +
<strong> 22aug</strong>
 +
</p>
 +
<p>
 +
<strong> Gibson assembly ,incubate 2h</strong>
 +
</p>
 +
<p>
 +
<strong> Run gel for colony pcr and frag 1+3 and the overnight cut of 3 in 1</strong>
 +
</p>
 +
<p>
 +
<strong>For colonies with correct band after colony pcr, pick colony from master plate, inoculate</strong>
 +
</p>
 +
<p>
 +
<strong>Frag 1+3, frag 2 re cut,</strong>
 +
</p>
 +
<p>
 +
<strong>Send mcyB to sequencing</strong>
 +
</p>
 +
<p>
 +
<strong>pick colony from plates  if have</strong>
 +
</p>
 +
<p>
 +
<strong>Transform gibson products</strong>
 +
</p>
 +
<p>
 +
<strong>Ligate frag 2 to 1+3</strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong>23 aug</strong>
 +
</p>
 +
<p>
 +
<strong>Run gel for Pcr dcas9 gfp,</strong>
 +
</p>
 +
<p>
 +
<strong>Run gel for Pcr 1+3 sgrna mcyI pet blue</strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong>26 aug</strong>
 +
</p>
 +
<p>
 +
<strong>Run gel for previous 40 colony pcr</strong>
 +
</p>
 +
<p>
 +
<strong>Inoculate L3 with correct band size</strong>
 +
</p>
 +
<p>
 +
<strong>Preparation for electroporation</strong>
 +
</p>
 +
<p>
 +
<strong>Transform ligation product and  3 in 1 for characterization</strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong>27 aug</strong>
 +
</p>
 +
<p>
 +
<strong>Pick clone for previous transformation</strong>
 +
</p>
 +
<p>
 +
<strong>3 in 1 plasmid pur using traditional method</strong>
 +
</p>
 +
<p>
 +
<strong>Re cut gib 5</strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong>28 aug</strong>
 +
</p>
 +
<p>
 +
<strong>PCR screening mcyI, B</strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong>29 aug</strong>
 +
</p>
 +
<p>
 +
<strong>Plasmid pur. Using tradition n takara</strong>
 +
</p>
 +
<p>
 +
<strong>Inoculate gib 5, prepare for measurement</strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong>30 aug</strong>
 +
</p>
 +
<p>
 +
<strong>I think no lab</strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong>31 aug</strong>
 +
</p>
 +
<p>
 +
<strong>Pick sgRNA clone?</strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong>1/9</strong>
 +
</p>
 +
<p>
 +
<strong>I think no lab</strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong>2/9</strong>
 +
</p>
 +
<p>
 +
<strong>I think no lab</strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong>3/9</strong>
 +
</p>
 +
<p>
 +
<strong>Re cut dcas9 gfp</strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong>4/9</strong>
 +
</p>
 +
<p>
 +
<strong>Run gel for re cut dcas9 gfp</strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong>5/9</strong>
 +
</p>
 +
<p>
 +
<strong>Transformation inoculate pet blue, puc19, 3 in 1, psb1c3 transformants</strong>
 +
</p>
 +
<p>
 +
<strong>Plasmid purification 3 in 1 using Takara plasmid purification kit</strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong>6/9</strong>
 +
</p>
 +
<p>
 +
<strong>Plasmid purification 3 in 1 using takara plasmid purification kit</strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong>9/9</strong>
 +
</p>
 +
<p>
 +
<strong>2 re cuts</strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong>10/9</strong>
 +
</p>
 +
<p>
 +
<strong>run gel for the colony pcr products (1.5 gel, all in) 65V 45 min 700 or no band</strong>
 +
</p>
 +
<p>
 +
<strong>Pick colony and colony pcr</strong>
 +
</p>
 +
<p>
 +
<strong>pour agar plate</strong>
 +
</p>
 +
<p>
 +
<strong> Take the 2 re cuts from 37C n run gel (1.5 gel 65V 45 min) shd be ~5500 and ~3500</strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong>11/9</strong>
 +
</p>
 +
<p>
 +
<strong>PCR clean for 3 in 1 cut</strong>
 +
</p>
 +
<p>
 +
<strong>Run gel for the dcas9 gfp sgrna no. 3 re cut for dcas9 gfp sgrna, cut the 5500 band (65V 45 min) (big well) (all in)</strong>
 +
</p>
 +
<p>
 +
<strong>Gel pur for the above cut gel (GM buffer 650ul)</strong>
 +
</p>
 +
<p>
 +
<strong>Run gel for colony pcr yesterday (1.5 gel)</strong>
 +
</p>
 +
<p>
 +
<strong>Set ligation mixture (re cuts of  3 in 1 and Mcy dcas9 gfp) 1:1</strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong>12/9</strong>
 +
</p>
 +
<p>
 +
<strong>Heat inactivate ligation</strong>
 +
</p>
 +
<p>
 +
<strong>2. Transform ligation (total dna 60ng)</strong>
 +
</p>
 +
<p>
 +
<strong>3. Dilute overnight inoculation, 1 to 3</strong>
 +
</p>
 +
<p>
 +
<strong>4. Set RE of 3 in 1 and sgrna dcas9 gfp</strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong>13/9</strong>
 +
</p>
 +
<p>
 +
<strong>Measure OD (growth curve) for modelling</strong>
 +
</p>
 +
<p>
 +
<strong>                                          </strong>
 +
</p>
 +
<p>
 +
<strong>16/9</strong>
 +
</p>
 +
<p>
 +
<strong>Re cuts for final product</strong>
 +
</p>
 +
<p>
 +
<strong>pick colony for growth curve</strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong>17/9</strong>
 +
</p>
 +
<p>
 +
<strong>growth curve</strong>
 +
</p>
 +
<p>
 +
<strong>Run gel for the RE cuts, pcr clean, start ligation</strong>
 +
</p>
 +
<p>
 +
<strong>transform ligation mixture</strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong>18/9</strong>
 +
</p>
 +
<p>
 +
<strong>Transofrmation of final ligation products</strong>
 +
</p>
 +
<p>
 +
<strong>2. OD measurements</strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong>19/9</strong>
 +
</p>
 +
<p>
 +
<strong> Pick colony (fat Clone)</strong>
 +
</p>
 +
<p>
 +
<strong>Transformation final product</strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong>20/9</strong>
 +
</p>
 +
<p>
 +
<strong>Pick clone</strong>
 +
</p>
 +
<p>
 +
<strong>Pcr check band</strong>
 +
</p>
 +
<p>
 +
 
 +
        <strong>·  Correct </strong>
 +
</p>
 +
<p>
 +
<strong>Plasmid pur by wch failed cuz me filp his gel..</strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong>23/9</strong>
 +
</p>
 +
<p>
 +
<strong>pick colony of final product</strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong>24/9</strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong>25/9</strong>
 +
</p>
 +
<p>
 +
<strong>Nanodrop final product</strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong>26/9</strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong>27/9</strong>
 +
</p>
 +
<p>
 +
<strong>run gel for the RE cuts and final product 24 and 15.</strong>
 +
</p>
 +
<p>
 +
<strong>All in for re cuts,</strong>
 +
</p>
 +
<p>
 +
<strong>5ul for final product 24 and 15</strong>
 +
</p>
 +
<p>
 +
<strong>65V 50min</strong>
 +
</p>
 +
<p>
 +
<strong>Nanodrop final product</strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong>30/9</strong>
 +
</p>
 +
<p>
 +
<strong>transformation</strong>
 +
</p>
 +
<p>
 +
<strong> final product plasmid 15 17 24 29(BL21)</strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong>1/10</strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong>2/10</strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong>3/10</strong>
 +
</p>
 +
<p>
 +
<strong>observe green fluorescence by uv exposure</strong>
 +
</p>
 +
<p>
 +
<strong>inoculate</strong>
 +
</p>
 +
<p>
 +
<strong>observe green fluorescence again by uv exposure</strong>
 +
</p>
 +
<p>
 +
 
 +
        <strong>·  All negative</strong>
 +
</p>
 +
<p>
 +
<strong>4/10</strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong>7/10</strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong>8/10</strong>
 +
</p>
 +
<p>
 +
<strong>Prepared 0.1mM NaCl, 0.1mM HEPES, 0.1mM Vitb12, all not sterilized. Picked 10 colonies, inoculated in 20mL Lb</strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong>10/10</strong>
 +
</p>
 +
<p>
 +
<strong>Plasmid purification with ethanol</strong>
 +
</p>
 +
<p>
 +
<strong>Filter b12</strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong>11/10</strong>
 +
</p>
 +
<p>
 +
<strong>OD (growth curve)</strong>
 +
</p>
 +
<p>
 +
<strong>Transformation of microcystis</strong>
 +
</p>
 +
<p>
 +
<strong>Electroporation</strong>
 +
</p>
 +
<p>
 +
<strong>Ligation by wch</strong>
 +
</p>
 +
<p>
 +
<strong>Dna purification</strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong>16/10</strong>
 +
</p>
 +
<p>
 +
<strong>Pour plate with kanamycin resistance and chl </strong>
 +
</p>
 +
<p>
 +
<strong>Plasmid Purification of Insert 1 and Insert 2</strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong>18/10</strong>
 +
</p>
 +
<p>
 +
<strong>Pick Insert 1 and 2 ,BL21 and NEB5a clones</strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
<p>
 +
<strong>Missing</strong>
 +
</p>
 +
<p>
 +
 
 +
        <strong>1.    20-21/7</strong>
 +
</p>
 +
<p>
 +
 
 +
        <strong>2.    23/7-8/8</strong>
 +
</p>
 +
<p>
 +
 
 +
        <strong>3.    24/9 (no gel pic n whatsapp)</strong>
 +
</p>
 +
<p>
 +
 
 +
        <strong>4.    26/9 (no gel pic n whatsapp)</strong>
 +
</p>
 +
<p>
 +
 
 +
        <strong>5.    1/10 (no gel pic n whatsapp)</strong>
 +
</p>
 +
<p>
 +
 
 +
        <strong>6.    2/10(no gel pic n whatsapp)</strong>
 +
</p>
 +
<p>
 +
 
 +
        <strong>7.    4/10(no gel pic n whatsapp)</strong>
 +
</p>
 +
<p>
 +
 
 +
        <strong>8.    7/10(no gel pic n whatsapp)</strong>
 +
</p>
 +
<p>
 +
<strong> </strong>
 +
</p>
 +
  
 
</html>
 
</html>

Revision as of 10:33, 21 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 Neb 5a

19 July

Picked 6 colonies

-Grow overnight in LB

Plasmid Purification (Takara)

-Colony 1 & 2 did not grow

-Run agarose gel 100V 30 mins

-Eluted DNA using 30uL elution buffer

-Nanodrop

22 July

Plasmid prep

Run gel for dcas9

Resuspend dna

23 July

1) Assembling frag1 +2 +3

-RE cut + ligation

NheI MliI BssSI-v2

2) dcas9 preparation Clean

3) Run gel Anson 5α

Transformed produce to Ecoli NEB5a

4) Cloned GFP to dCas9

-RE cut + ligation

EcoRI NsiI

Transformed product to Ecoli NEB5a

5) Transformed PUC19 from stock (for cloning sgRNA later)

1 Aug

Cyanobac arrive, cultured in 15x4 mL medium

2 Aug

6 Aug

Colony PCR

PCR Clean, RE ligation

7 Aug

-gel purification after 75V 45min 0.7 gel

- Gibson assembly

- Run Gel for Overnigt SgRNA PCR product

- RE(2hrs incubation)+ligation for SgRNA and pETBlue

-Transformed 3in1, pETBlue sgRNA and L3 into NEB5a

8 Aug

Colony PCR, run gel, pick clone

9 August

1) Checked plates for sgRNA pet blue mcyI

sgRNA pet blue mcyI 2

sgRNA pet blue mcyB 2

3 in 1

Gib 2.1

Gib 2.2

-All no colony.

2) Conducted PCR purification of overnight (14h) RE cut of dCas9 GFP

-50rxn mixture

3) Conducted ligation

1:3 & 1:5

Vector 250ng

Both 20uL rxn,

-rm temp for 10 min

Heat inactive at 65°C for 10 min

On ice 90min

-Then add ligase again

Ligate over weekend.

4) Amplified frag1 + 2 + 3

-sgRNA I, sgRNA B, GFP

For Gibson

Frag 1 2 3 gfp, 65°C

sgRNAs 55C

All 50 uL rxn

5) Weight powder, for SOC (800mL no glucose/ mgXX) (no yellow)

-LB agar (have yellow) 1800uL

Glucose 9g

6) Run gel for all 6 frags (all in)

-75V 45 mins

Ladder use 1kb & 50bp

Loading dye 6X

7) Gel Purification -elude using 30uL elution b.

12 August

1) Colony PCR for those without bands reaction

-Picked colonies from master plate

2) Performed Gibson(Gib3) & NEBuilder

- Scaled down to 10mL rxn

- Concentration acc to following nanodrop

-incubation 1h

3) PCR for XGib 1 (frag1) Gib1 (frag1)

XGib 2 (frag2) Gib2 (frag2)

XGib 3 (frag3) Gib3 (frag3)

4) Colony PCR no.24 correct band

(dcas9 gfp) L3

Band size 700-800 bp

Inoculated 24 (picked from master plate)

5) 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

2) Checked plates from 12/8

Colony: petblue +ve

dCas9 gfp 1:3

dCas9 gfp 1:5

3) Plasmid purification L3

dCas9 gfp 24

Re Cut for frag1-3 (gib)

McyI, McyB

14 August

1) Run Gel for X gib Frag 1

-No bands at all

Possibly due to pipetting errors.

2) Started 10uL reaction using Gibson concentration according to following gel. (pic Gib4)

Reaction: Frag3: 50ng

Frag2: 38.73ng

Frag1: 29.26ng

Nebuilder: 5uL

H2O

3) Set up PCR reaction for XGib Frag2

XGib Frag3

98°C 98°C 66° 72°C 72°C 4°C
30sec 5s 15s 1.5min 2min ¥

4) Transformed Gib 4.1 Gib 4.2

Overnight ligation sgRNA mcyI Petblue

sgRNA mcyB petblue

Gib -0.9uL DNA

sgRNA -2uL

5) Checked plates for previous transformation: Gib 3 in 1 -

Gib 2 - No colonies

Gib 3 -

From last yr -petblue stock -yes colonies

6) Picked 6 colonies from petblue

Petblue 1-3 -3mL (with

Petblue 4-5 -2mL amp)

Master plate -No xgal Iptg

7) Run gel for pcr products

Frag 2, frag 3 X Gib

Gel purification

DNA concentration on 13/8

15 August

1) RE cut for frag1 frag 2 and frag 3

X Gib ~6 hr

Nanodrop before RE cut

2) Set up Gibson reaction

a. Frag 3 50nL 16uL -

b. Frag 2 38.73ng 2.1 uL -

c. Frag 1 29.26ng 0.4uL - 20uL/xh

d. Nebuilder 10uL -

e. H2O 5.9uL -

50C for 2hr

3) Plasmid Purification of Petblue

Elude using 30uL

Nanodrop

4) Gib 4.1 -Petblue mcyB plasmid have 1 colony each

-Picked & inoculated

5) PCR purified RE cuts from (nolning?)

-ligated H3 & 3 in 1

1+3 1:1

1+2+3 1:1:1

6) 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.

2) Run gel for frag 1+3 & 3in1 (ligated)

3) Intended to amp 1+3 using 3F + 1R and 3R + 1F

4) 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

5) 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

2) Inoculated 2 3mL tubes of Gib4

-Gib4.1 & 4.2

From master plate

3) Set up overnight PCR to amp mcyI, frag1, frag3 xgib,

4) RE cut mcyI (247.8ng), petblue (1000ng), frag1 (741ng)- frag3 (140ng) overnight [McyI only added 3uL]

5) Checked Neb 3 in 1 using different primers

Checked frag 1+3

20 August

1) Run gel for mcyI, frag1-3 xGib

-gel purification Takara

2) PCR clean mcyI, petblue 2 from overnight RE

3) Ligated mcyI petblue, frag1+3

-vector 100ng -vector 100ng

1:3 1:1

4) ?

5) Plasmid pur Gib6.4

-yield extremely low

6) 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

2) Collected overnight ligation

-Amplified frag 1+3 wing

Primer frag1 F & primer frag3 R (vice versa also)

3) Transformed sgRNA mcyI petblue and dCas9 gfp (L3)

4) Received sanger sequence results

-99.4% identical

Mutation- X

5) Picked 25 colonies from previous L3 plate

-colony PCR 25-49 -one Taq

50 - sapphire

6) Single cut & double cut 3 in 1 plasmid

-All undesired results

22aug

Gibson assembly ,incubate 2h

Run gel for colony pcr and frag 1+3 and the overnight cut of 3 in 1

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

Frag 1+3, frag 2 re cut,

Send mcyB to sequencing

pick colony from plates if have

Transform gibson products

Ligate frag 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

Re cut gib 5

28 aug

PCR screening mcyI, B

29 aug

Plasmid pur. Using tradition n takara

Inoculate gib 5, prepare for measurement

30 aug

I think no lab

31 aug

Pick sgRNA clone?

1/9

I think no lab

2/9

I think no lab

3/9

Re cut dcas9 gfp

4/9

Run gel for re 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 re cuts

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 re cuts from 37C n run gel (1.5 gel 65V 45 min) shd be ~5500 and ~3500

11/9

PCR clean for 3 in 1 cut

Run gel for the dcas9 gfp sgrna no. 3 re cut for dcas9 gfp sgrna, cut the 5500 band (65V 45 min) (big well) (all in)

Gel pur for the above cut gel (GM buffer 650ul)

Run gel for colony pcr yesterday (1.5 gel)

Set ligation mixture (re 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 RE of 3 in 1 and sgrna dcas9 gfp

13/9

Measure OD (growth curve) for modelling

16/9

Re cuts for final product

pick colony for growth curve

17/9

growth curve

Run gel for the RE 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 pur by wch failed cuz me filp his gel..

23/9

pick colony of final product

24/9

25/9

Nanodrop final product

26/9

27/9

run gel for the RE cuts and final product 24 and 15.

All in for re cuts,

5ul for final product 24 and 15

65V 50min

Nanodrop final product

30/9

transformation

final product plasmid 15 17 24 29(BL21)

1/10

2/10

3/10

observe green fluorescence by uv exposure

inoculate

observe green fluorescence again by uv exposure

· All negative

4/10

7/10

8/10

Prepared 0.1mM NaCl, 0.1mM HEPES, 0.1mM Vitb12, all not sterilized. Picked 10 colonies, inoculated in 20mL Lb

10/10

Plasmid purification with ethanol

Filter b12

11/10

OD (growth curve)

Transformation of microcystis

Electroporation

Ligation by wch

Dna purification

16/10

Pour plate with kanamycin resistance and chl

Plasmid Purification of Insert 1 and Insert 2

18/10

Pick Insert 1 and 2 ,BL21 and NEB5a clones

Missing

1. 20-21/7

2. 23/7-8/8

3. 24/9 (no gel pic n whatsapp)

4. 26/9 (no gel pic n whatsapp)

5. 1/10 (no gel pic n whatsapp)

6. 2/10(no gel pic n whatsapp)

7. 4/10(no gel pic n whatsapp)

8. 7/10(no gel pic n whatsapp)