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

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<html>
 
<html>
  
 +
<p>
 +
17 July
 +
</p>
 +
<p>
 +
Transformation of dcas9 plasmid 2019 kit plate 5 well 22n.   
 +
</p>
 +
<p>
 +
- kanamycin resistance plate
 +
</p>
 +
<p>
 +
- to E. coli NEB5a
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
19 July
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
Picked 6 colonies
 +
</p>
 +
<p>
 +
-grow overnight in LB
 +
</p>
 +
<p>
 +
Plasmid purification (Takara)
 +
</p>
 +
<p>
 +
-colony 1 & 2 did not grow
 +
</p>
 +
<p>
 +
-run agarose gel for 100v 30 mins
 +
</p>
 +
<p>
 +
-eluted DNA using 30ul elution buffer
 +
</p>
 +
<p>
 +
-nanodrop the eluted DNA
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
22 July
 +
</p>
 +
<p>
 +
Plasmid prep 
 +
</p>
 +
<p>
 +
Run gel for dCas9
 +
</p>
 +
<p>
 +
Resuspend DNA
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
23 July
 +
</p>
 +
<ol>
  
−
<div class="column full_size">
+
<li>Assembling Fragment 1+2+3
 +
</li>
 +
</ol>
 +
<p>
 +
-restriction endonuclease cut + ligation
 +
</p>
 +
<p>
 +
NheI and BssSI-v2
 +
</p>
 +
<p>
 +
 +
</p>
 +
<ol>
  
−
<h1>Notebook</h1>
+
<li>Dcas9 preparation clean
−
<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>
+
  
−
</div>
+
<li>Run gel  NEB5α
−
<div class="clear"></div>
+
</li>
 +
</ol>
 +
<p>
 +
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
Transformed produce to E.coli neb5
 +
</p>
 +
<p>
 +
 +
</p>
 +
<ol>
  
 +
<li>Cloned GFP to dcas9
 +
</li>
 +
</ol>
 +
<p>
 +
-restriction endonuclease cut + ligation
 +
</p>
 +
<p>
 +
EcorI NsiI
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
Transformed product to E.coli neb5
 +
</p>
 +
<p>
 +
 +
</p>
 +
<ol>
  
 +
<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>
  
−
<div class="column two_thirds_size">
+
<li>Checked plates for sgrna pet blue mcyi
−
<h3>What should this page have?</h3>
+
</li>
−
<ul>
+
</ol>
−
<li>Chronological notes of what your team is doing.</li>
+
<p>
−
<li> Brief descriptions of daily important events.</li>
+
Sgrna pet blue mcyI2
−
<li>Pictures of your progress. </li>
+
</p>
−
<li>Mention who participated in what task.</li>
+
<p>
−
</ul>
+
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>
  
−
</div>
+
<li>Conducted PCR purification of overnight (14h) restriction endonuclease cut of dcas9 gfp (EcoRI, NsiI)
 +
</li>
 +
</ol>
 +
<p>
 +
-50 rxn mixture
 +
</p>
 +
<ol>
  
−
<div class="column third_size">
+
<li>Conducted ligation
−
<div class="highlight decoration_A_full">
+
</li>
−
<h3>Inspiration</h3>
+
</ol>
−
<p>You can see what others teams have done to organize their notes:</p>
+
<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>
  
−
<ul>  
+
<li>Amplified Fragment1 + 2 + 3
−
<li><a href="https://2018.igem.org/Team:Munich/Notebook">2018 Munich</a></li>
+
</li>
−
<li><a href="https://2014.igem.org/Team:ATOMS-Turkiye/Notebook">2014 ATOMS-Turkiye</a></li>
+
</ol>
−
<li><a href="https://2014.igem.org/Team:Tec-Monterrey/ITESM14_project.html#tab_notebook">2014 Tec Monterrey</a></li>
+
<p>
−
<li><a href="https://2014.igem.org/Team:Kyoto/Notebook/Magnetosome_Formation#title">2014 Kyoto</a></li>
+
-sgrna I, sgrna B, gfp
−
<li><a href="https://2014.igem.org/Team:Cornell/notebook">2014 Cornell</a></li>
+
</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>
 +
 
 +
<li>Weight powder, for soc (800ml no glucose/ mgxx) (no yellow)
 +
</li>
 +
</ol>
 +
<p>
 +
-LB agar (have yellow) 1800ul
 +
</p>
 +
<p>
 +
Glucose 9g
 +
</p>
 +
<p>
 +
 +
</p>
 +
<ol>
 +
 
 +
<li>Run gel for all 6 Fragments (all in)
 +
</li>
 +
</ol>
 +
<p>
 +
-75v 45 mins
 +
</p>
 +
<p>
 +
Ladder use 1kb & 50bp
 +
</p>
 +
<p>
 +
Loading dye 6x
 +
</p>
 +
<p>
 +
 +
</p>
 +
<ol>
 +
 
 +
<li>Gel purification -elude using 30ul elution b.  
 +
</li>
 +
</ol>
 +
<p>
 +
 +
</p>
 +
<p>
 +
12 August
 +
</p>
 +
<p>
 +
 +
</p>
 +
<ol>
 +
 
 +
<li>Colony PCR for those without bands reaction 
 +
</li>
 +
</ol>
 +
<p>
 +
-picked colonies from master plate
 +
</p>
 +
<p>
 +
 +
</p>
 +
<ol>
 +
 
 +
<li>Performed gibson(gib3) & nebuilder
 +
 
 +
<li>Scaled down to 10ml rxn
 +
 
 +
<li>Concentration acc to following nanodrop
 +
</li>
 +
</ol>
 +
<p>
 +
-incubation 1h
 +
</p>
 +
<p>
 +
 +
</p>
 +
<ol>
 +
 
 +
<li>PCR for xgib 1 (Fragment1) gib1 (Fragment1)
 +
</li>
 +
</ol>
 +
<p>
 +
            xgib 2 (Fragment2) gib2 (Fragment2)
 +
</p>
 +
<p>
 +
xgib 3 (Fragment3) gib3 (Fragment3)
 +
</p>
 +
<p>
 +
 +
</p>
 +
<ol>
 +
 
 +
<li>Colony PCR no.24 correct band
 +
</li>
 +
</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>
 +
 
 +
<li>Transformed gibson 1 gibson 2 gibson 3.2  nebuilder (1)
 +
</li>
 +
</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>
 +
 
 +
<li>Run gel for previous PCR 
 +
</li>
 +
</ol>
 +
<p>
 +
 +
</p>
 +
<ol>
 +
 
 +
<li>Checked plates from 12/8
 +
</li>
 +
</ol>
 +
<p>
 +
Colony: petblue +ve
 +
</p>
 +
<p>
 +
dCas9 gfp 1:3
 +
</p>
 +
<p>
 +
dCas9 gfp 1:5
 +
</p>
 +
<p>
 +
 +
</p>
 +
<ol>
 +
 
 +
<li>Plasmid purification l3
 +
</li>
 +
</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>
 +
 
 +
<li>Run gel for xgib Fragment 1
 +
</li>
 +
</ol>
 +
<p>
 +
-no bands at all
 +
</p>
 +
<p>
 +
Possibly due to pipetting errors.  
 +
</p>
 +
<p>
 +
 +
</p>
 +
<ol>
 +
 
 +
<li>Started 10ul reaction using gibson concentration according to following gel. (pic gib4)
 +
</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>
 +
 
 +
<li>Set up PCR reaction for xgib Fragment2
 +
</li>
 +
</ol>
 +
<p>
 +
Xgib Fragment3
 +
</p>
 +
<p>
 +
 +
</p>
 +
 
 +
<table>
 +
  <tr>
 +
  <td>98c
 +
  </td>
 +
  <td>98c
 +
  </td>
 +
  <td>66c
 +
  </td>
 +
  <td>72c
 +
  </td>
 +
  <td>72c
 +
  </td>
 +
  <td>4c
 +
  </td>
 +
  </tr>
 +
  <tr>
 +
  <td>30sec
 +
  </td>
 +
  <td>5s
 +
  </td>
 +
  <td>15s
 +
  </td>
 +
  <td>1.5min
 +
  </td>
 +
  <td>2min
 +
  </td>
 +
  <td>
 +
  </td>
 +
  </tr>
 +
</table>
 +
 
 +
 
 +
<p>
 +
 +
</p>
 +
<p>
 +
 +
</p>
 +
<ol>
 +
 
 +
<li>Transformed gib 4.1 gib 4.2
 +
</li>
 +
</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>
 +
 
 +
<li>Checked plates for previous transformation: gib 3 in 1 -
 +
</li>
 +
</ol>
 +
<p>
 +
  gib 2    - no colonies
 +
</p>
 +
<p>
 +
  gib 3    - from last year -PETblue stock -yes colonies
 +
</p>
 +
<p>
 +
 +
</p>
 +
<ol>
 +
 
 +
<li>Picked 6 colonies from petblue
 +
</li>
 +
</ol>
 +
<p>
 +
petblue 1-3 -3ml(with amp)
 +
</p>
 +
<p>
 +
petblue 4-5 -2ml       
 +
</p>
 +
<p>
 +
Master plate -no xgal iptg
 +
</p>
 +
<p>
 +
 +
</p>
 +
<ol>
 +
 
 +
<li>Run gel for PCR products 
 +
</li>
 +
</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>
 +
 
 +
<li>Restriction endonuclease cut for Fragment1 Fragment 2 and Fragment 3
 +
</li>
 +
</ol>
 +
<p>
 +
Xgib ~6 hr (MluI, BssaI and NheI)
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
nanodrop before restriction endonuclease cut
 +
</p>
 +
<p>
 +
 +
</p>
 +
<ol>
 +
 
 +
<li>Set up gibson reaction
 +
 
 +
<li>Fragment 3 50ng      16ul-
 +
 
 +
<li>Fragment 2 38.73ng 2.1 ul-
 +
 
 +
<li>Fragment 1 29.26ng 0.4ul-20ul/xh
 +
 
 +
<li>Nebuilder                10ul-
 +
 
 +
<li>H2o                      5.9ul-
 +
</li>
 +
</ol>
 +
<p>
 +
 +
</p>
 +
<p>
 +
50c for 2hr (optimized protocol)
 +
</p>
 +
<p>
 +
 +
</p>
 +
<ol>
 +
 
 +
<li>Plasmid purification of petblue 
 +
</li>
 +
</ol>
 +
<p>
 +
            elute using 30ul 
 +
</p>
 +
<p>
 +
nanodrop
 +
</p>
 +
<p>
 +
 +
</p>
 +
<ol>
 +
 
 +
<li>Gib 4.1 -petblue mcyb plasmid have 1 colony each
 +
</li>
 +
</ol>
 +
<p>
 +
-picked & inoculated
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
 +
</p>
 +
<ol>
 +
 
 +
<li>PCR purified restriction endonuclease cuts from (Fragment 1, 2, 3 13/8?)
 +
</li>
 +
</ol>
 +
<p>
 +
-ligated h3 & 3 in 1
 +
</p>
 +
<p>
 +
1+3 1:1
 +
</p>
 +
<p>
 +
1+2+31:1:1
 +
</p>
 +
<p>
 +
 +
</p>
 +
<ol>
 +
 
 +
<li>Sequencing
 +
</li>
 +
</ol>
 +
<p>
 +
 +
</p>
 +
<p>
 +
16 August
 +
</p>
 +
<p>
 +
 +
</p>
 +
<ol>
 +
 
 +
<li>Plasmid purification of 
 +
</li>
 +
</ol>
 +
<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>
 +
 
 +
<li>Run gel for Fragment 1+3 & 3in1 (ligated)
 +
</li>
 +
</ol>
 +
<p>
 +
 +
</p>
 +
<ol>
 +
 
 +
<li>Intended to amp 1+3 using 3f + 1r and 3r + 1f
 +
</li>
 +
</ol>
 +
<p>
 +
 +
</p>
 +
<ol>
 +
 
 +
<li>Intended to check gib4 using 1+2, 2+3, 1+3 primers.
 +
</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>
 +
 
 +
<li>Intended to check sgrna mcyb petblue using colony PCR
 +
</li>
 +
</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>
 +
 
 +
<li>Colony PCR for mcyb sgrna petblue
 +
</li>
 +
</ol>
 +
<p>
 +
(only 1 white colony)
 +
</p>
 +
<p>
 +
Results positive: band size ~300bp
 +
</p>
 +
<p>
 +
 +
</p>
 +
<ol>
 +
 
 +
<li>Inoculated 2 3ml tubes of gib4
 +
</li>
 +
</ol>
 +
<p>
 +
-gib4.1 & 4.2
 +
</p>
 +
<p>
 +
From master plate
 +
</p>
 +
<p>
 +
 +
</p>
 +
<ol>
 +
 
 +
<li>Set up overnight PCR to amp mcyi, Fragment1, Fragment3 xgib,
 +
</li>
 +
</ol>
 +
<p>
 +
 +
</p>
 +
<ol>
 +
 
 +
<li>Restriction endonuclease (PstI, HindIII, MluI, BssaI, NheI) cut mcyi (247.8ng), petblue (1000ng), Fragment1 (741ng)- Fragment3 (140ng) overnight [mcyi only added 3ul]
 +
</li>
 +
</ol>
 +
<p>
 +
 +
</p>
 +
<ol>
 +
 
 +
<li>Checked neb 3 in 1 using different primers 
 +
</li>
 +
</ol>
 +
<p>
 +
Checked Fragment 1+3
 +
</p>
 +
<p>
 +
 +
</p>
 +
<p>
 +
20 August 
 +
</p>
 +
<p>
 +
 +
</p>
 +
<ol>
 +
 
 +
<li>Run gel for mcyi, Fragment1-3 xgib
 +
</li>
 +
</ol>
 +
<p>
 +
-gel purification takara
 +
</p>
 +
<p>
 +
 +
</p>
 +
<ol>
 +
 
 +
<li>PCR clean mcyi, petblue 2 from overnight restriction endonuclease cut
 +
</li>
 +
</ol>
 +
<p>
 +
 +
</p>
 +
<ol>
 +
 
 +
<li>Ligated mcyi petblue, Fragment1+3
 +
</li>
 +
</ol>
 +
<p>
 +
-vector 100ng-vector 100ng
 +
</p>
 +
<p>
 +
1:3 1:1
 +
</p>
 +
<p>
 +
 +
</p>
 +
<ol>
 +
 
 +
<li>?
 +
</li>
 +
</ol>
 +
<p>
 +
 +
</p>
 +
<ol>
 +
 
 +
<li>Plasmid purification gib6.4
 +
</li>
 +
</ol>
 +
<p>
 +
-yield extremely low
 +
</p>
 +
<p>
 +
 +
</p>
 +
<ol>
 +
 
 +
<li>Picked colonies: 2 nebuilder 3in1.-
 +
</li>
 +
</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>
 +
 
 +
<li>Plasmid pur for 6 tubes -nebuilder x2, gib4 x2, sgrna mcyb petblue
 +
</li>
 +
</ol>
 +
<p>
 +
 +
</p>
 +
<ol>
 +
 
 +
<li>Collected overnight ligation
 +
</li>
 +
</ol>
 +
<p>
 +
-amplified Fragment 1+3 wing
 +
</p>
 +
<p>
 +
Primer Fragment1 f & primer Fragment3 r (vice versa also)
 +
</p>
 +
<p>
 +
 +
</p>
 +
<ol>
 +
 
 +
<li>Transformed sgrna mcyi petblue and dcas9 gfp (l3)
 +
</li>
 +
</ol>
 +
<p>
 +
 +
</p>
 +
<ol>
 +
 
 +
<li>Received sanger sequence results 
 +
</li>
 +
</ol>
 +
<p>
 +
-99.4% identical
 +
</p>
 +
<p>
 +
mutation- x
 +
</p>
 +
<p>
 +
 +
</p>
 +
<ol>
 +
 
 +
<li>Picked 25 colonies from previous l3 plate
 +
</li>
 +
</ol>
 +
<p>
 +
-colony PCR 25-49  -one taq
 +
</p>
 +
<p>
 +
50- sapphire
 +
</p>
 +
<p>
 +
 +
</p>
 +
<ol>
 +
 
 +
<li>Single cut & double cut 3 in 1 plasmid
 +
</li>
 +
</ol>
 +
<p>
 +
-all undesired results
 +
</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>
 +
 
 +
<li>Correct 
 +
</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>
 
</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>
−
</div>
+
 
 +
</body>
  
 
</html>
 
</html>

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