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

 
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<html>
 
<html>
  
−
 
−
<div class="column full_size">
 
 
<p>
 
<p>
−
<strong>17 July</strong>
+
17 July  
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>Transformation of dCas9 plasmid 2019 kit plate 5 well 22N.</strong>
+
Transformation of dcas9 plasmid 2019 kit plate 5 well 22n.    
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>- Kanamycin Resistance plate</strong>
+
- kanamycin resistance plate  
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>- to E. Coli Neb 5a  </strong>
+
- to E. coli NEB5a
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>19 July</strong>
+
19 July  
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>Picked 6 colonies</strong>
+
Picked 6 colonies  
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>-Grow overnight in LB</strong>
+
-grow overnight in LB  
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>Plasmid Purification (Takara)</strong>
+
Plasmid purification (Takara)  
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>-Colony 1 & 2 did not grow</strong>
+
-colony 1 & 2 did not grow  
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>-Run agarose gel 100V 30 mins</strong>
+
-run agarose gel for 100v 30 mins  
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>-Eluted DNA using 30uL elution buffer</strong>
+
-eluted DNA using 30ul elution buffer  
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>  -Nanodrop      </strong>
+
-nanodrop the eluted DNA
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>22 July</strong>
+
22 July  
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>Plasmid prep</strong>
+
Plasmid prep
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>Run gel for dcas9</strong>
+
Run gel for dCas9
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>Resuspend dna</strong>
+
Resuspend DNA
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>23 July</strong>
+
23 July  
 
</p>
 
</p>
−
<p>
+
<ol>
  
−
        <strong>1) Assembling frag1 +2 +3</strong>
+
<li>Assembling Fragment 1+2+3  
−
</p>
+
</li>
 +
</ol>
 
<p>
 
<p>
−
 
+
-restriction endonuclease cut + ligation  
−
    <strong>-RE cut + ligation</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
NheI and BssSI-v2  
−
    <strong>NheI MliI BssSI-v2</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
 +
 +
</p>
 +
<ol>
  
−
    <strong> </strong>
+
<li>Dcas9 preparation clean
 +
 
 +
<li>Run gel  NEB5α
 +
</li>
 +
</ol>
 +
<p>
 +
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
−
        <strong>2) dcas9 preparation Clean</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
Transformed produce to E.coli neb5
−
        <strong>3) Run gel Anson 5α</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
 +
 +
</p>
 +
<ol>
  
−
    <strong> </strong>
+
<li>Cloned GFP to dcas9
 +
</li>
 +
</ol>
 +
<p>
 +
-restriction endonuclease cut + ligation
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
EcorI NsiI
−
    <strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>Transformed produce to Ecoli NEB5a</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
Transformed product to E.coli neb5
 
</p>
 
</p>
 
<p>
 
<p>
 +
 +
</p>
 +
<ol>
  
−
        <strong>4) Cloned GFP to dCas9</strong>
+
<li>Transformed puc19 from stock (for cloning sgrna later)  
 +
</li>
 +
</ol>
 +
<p>
 +
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
−
    <strong>-RE cut + ligation</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
−
    <strong>EcoRI NsiI</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
−
    <strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
1 Aug
−
    <strong>Transformed product to Ecoli NEB5a</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
Cyanobacteria arrived, cultured in 15x4 ml medium
−
    <strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
GFP-dcas9 plasmid ligation (all failed)  
−
        <strong>5) Transformed PUC19 from stock (for cloning sgRNA later)</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
2 Aug
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
Colony PCR for l2, 3, 4
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>1 Aug</strong>
+
PCR Fragment 1, 2, 3
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>Cyanobac arrive, cultured in 15x4 mL medium</strong>
+
Plasmid purification
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>2 Aug</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
6 Aug
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
-colony PCR 
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>6 Aug</strong>
+
-standard q5 PCR preperation for gibson 
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>Colony PCR</strong>
+
-gibson
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>PCR Clean, RE ligation</strong>
+
- gfp+ dcas9 plasmid again as l3
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>7 Aug</strong>
+
 
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>-gel purification after 75V 45min 0.7 gel</strong>
+
7 Aug
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>- Gibson assembly</strong>
+
-gel purification after 75v 45min 0.7 gel 
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>- Run Gel for Overnigt SgRNA PCR product</strong>
+
- gibson assembly 
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>- RE(2hrs incubation)+ligation for SgRNA and pETBlue</strong>
+
- run gel for overnight sgrna PCR product 
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>-Transformed 3in1, pETBlue sgRNA and L3 into NEB5a</strong>
+
- restriction endonuclease cut(PstI, HindIII)(2hrs incubation)+ligation for sgrna and petblue 
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
-transformed 3in1, petblue sgrna and L3 into NEB5a
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>8 Aug</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>Colony PCR, run gel, pick clone</strong>
+
8 Aug
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
Colony PCR for l3 
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>9 August</strong>
+
Run gel for 3in1 (1 smear), 
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
Pick clone
−
        <strong>1) Checked plates for sgRNA pet blue mcyI</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
−
        <strong>sgRNA pet blue mcyI 2</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
 +
9 August
 +
</p>
 +
<ol>
  
−
    <strong>sgRNA pet blue mcyB 2</strong>
+
<li>Checked plates for sgrna pet blue mcyi
 +
</li>
 +
</ol>
 +
<p>
 +
Sgrna pet blue mcyI2
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
Sgrna pet blue mcyb 2
−
    <strong>3 in 1</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
3 in 1  
−
    <strong>Gib 2.1</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
Gib 2.1
−
    <strong>Gib 2.2</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
Gib 2.2
−
    <strong>-All no colony.</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
 +
-all no colony.
 +
</p>
 +
<ol>
  
−
        <strong>2) Conducted PCR purification of overnight (14h) RE cut of dCas9 GFP</strong>
+
<li>Conducted PCR purification of overnight (14h) restriction endonuclease cut of dcas9 gfp (EcoRI, NsiI)
 +
</li>
 +
</ol>
 +
<p>
 +
-50 rxn mixture
 
</p>
 
</p>
 +
<ol>
 +
 +
<li>Conducted ligation
 +
</li>
 +
</ol>
 
<p>
 
<p>
−
 
+
1:3 & 1:5
−
    <strong>-50rxn mixture</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
Vector 250ng
−
        <strong>3) Conducted ligation</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
Both 20ul rxn,
−
    <strong>1:3 & 1:5</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
−
    <strong>Vector 250ng</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
-rm temp for 10 min
−
    <strong>Both 20uL rxn,</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
heat inactive at 65c for 10 min
−
    <strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
on ice 90min
−
    <strong>-rm temp for 10 min</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
-then add ligase again
−
    <strong>        Heat inactive at 65°C for 10 min</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
ligate over weekend.
−
    <strong>        On ice 90min</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>-Then add ligase again</strong>
+
 
</p>
 
</p>
 +
<ol>
 +
 +
<li>Amplified Fragment1 + 2 + 3
 +
</li>
 +
</ol>
 
<p>
 
<p>
−
<strong>        Ligate over weekend.</strong>
+
-sgrna I, sgrna B, gfp
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
For gibson
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
Fragment 1 2 3 gfp, 65c
−
        <strong>4) Amplified frag1 + 2 + 3</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
Sgrnas 55c
−
    <strong>-sgRNA I, sgRNA B, GFP</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
−
        <strong>For Gibson</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
All 50 ul rxn
−
    <strong>Frag 1 2 3 gfp, 65°C</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
 +
 +
</p>
 +
<ol>
  
−
    <strong>sgRNAs 55C</strong>
+
<li>Weight powder, for soc (800ml no glucose/ mgxx) (no yellow)
 +
</li>
 +
</ol>
 +
<p>
 +
-LB agar (have yellow) 1800ul
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
Glucose 9g
−
    <strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
 +
 +
</p>
 +
<ol>
  
−
        <strong>All 50 uL rxn</strong>
+
<li>Run gel for all 6 Fragments (all in)
 +
</li>
 +
</ol>
 +
<p>
 +
-75v 45 mins
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
Ladder use 1kb & 50bp
−
    <strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
Loading dye 6x
−
        <strong>5) Weight powder, for SOC (800mL no glucose/ mgXX) (no yellow)</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
−
    <strong>-LB agar (have yellow) 1800uL</strong>
+
 
</p>
 
</p>
−
<p>
+
<ol>
  
−
    <strong>Glucose 9g</strong>
+
<li>Gel purification -elude using 30ul elution b.
−
</p>
+
</li>
 +
</ol>
 
<p>
 
<p>
−
 
+
−
    <strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
12 August
−
        <strong>6) Run gel for all 6 frags (all in)</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
−
    <strong>-75V 45 mins</strong>
+
 
</p>
 
</p>
−
<p>
+
<ol>
  
−
    <strong>Ladder use 1kb & 50bp</strong>
+
<li>Colony PCR for those without bands reaction 
−
</p>
+
</li>
 +
</ol>
 
<p>
 
<p>
−
 
+
-picked colonies from master plate
−
    <strong>Loading dye 6X</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
 +
 +
</p>
 +
<ol>
  
−
    <strong> </strong>
+
<li>Performed gibson(gib3) & nebuilder
−
</p>
+
−
<p>
+
  
−
        <strong>7) Gel Purification -elude using 30uL elution b.</strong>
+
<li>Scaled down to 10ml rxn
−
</p>
+
 
 +
<li>Concentration acc to following nanodrop
 +
</li>
 +
</ol>
 
<p>
 
<p>
−
<strong>12 August</strong>
+
-incubation 1h
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
−
<p>
+
<ol>
  
−
        <strong>1) Colony PCR for those without bands reaction</strong>
+
<li>PCR for xgib 1 (Fragment1) gib1 (Fragment1)
 +
</li>
 +
</ol>
 +
<p>
 +
            xgib 2 (Fragment2) gib2 (Fragment2)
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
xgib 3 (Fragment3) gib3 (Fragment3)
−
    <strong>-Picked colonies from master plate</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
−
    <strong> </strong>
+
 
</p>
 
</p>
−
<p>
+
<ol>
  
−
        <strong>2) Performed Gibson(Gib3) & NEBuilder</strong>
+
<li>Colony PCR no.24 correct band
−
</p>
+
</li>
 +
</ol>
 
<p>
 
<p>
−
 
+
(dcas9 gfp) L3
−
        <strong>-      Scaled down to 10mL rxn</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
band size 700-800 bp
−
        <strong>-       Concentration acc to following nanodrop</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
            inoculated 24 Hours (picked from master plate)
−
    <strong>-incubation 1h</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
−
<p>
+
<ol>
  
−
        <strong>3) PCR for XGib 1 (frag1) Gib1 (frag1)</strong>
+
<li>Transformed gibson 1 gibson 2 gibson 3.2  nebuilder (1)  
−
</p>
+
</li>
 +
</ol>
 
<p>
 
<p>
−
<strong>                    XGib 2 (frag2) Gib2 (frag2)</strong>
+
+ve control  petblue nebuilder (2)  
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>                    XGib 3 (frag3) Gib3 (frag3)</strong>
+
      dcas9 gfp 1:3  
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
      dcas9 gfp 1:5
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
          gibson 3.1
−
        <strong>4) Colony PCR no.24 correct band</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
−
        <strong>(dcas9 gfp) L3</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>        Band size 700-800 bp</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>        Inoculated 24 (picked from master plate)</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
−
        <strong>5) Transformed Gibson 1</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>                          Gibson 2</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>        Gibson 3.2 NEBuilder (1)</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>        +ve control  petblue NEBuilder (2)</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>                          dCas9 gfp 1:3</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>                          dCas9 gfp 1:5</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>                          Gibson 3.1</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>13 August</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
13 August
−
        <strong>1) Run gel for previous PCR</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
 +
<ol>
 +
 +
<li>Run gel for previous PCR 
 +
</li>
 +
</ol>
 
<p>
 
<p>
−
 
+
−
        <strong>2) Checked plates from 12/8</strong>
+
 
</p>
 
</p>
−
<p>
+
<ol>
  
−
    <strong>Colony: petblue +ve</strong>
+
<li>Checked plates from 12/8
−
</p>
+
</li>
 +
</ol>
 
<p>
 
<p>
−
 
+
Colony: petblue +ve
−
    <strong>        dCas9 gfp 1:3</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
dCas9 gfp 1:3
−
    <strong>        dCas9 gfp 1:5</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
dCas9 gfp 1:5
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
−
        <strong>3) Plasmid purification L3</strong>
+
 
</p>
 
</p>
−
<p>
+
<ol>
  
−
    <strong>dCas9 gfp 24</strong>
+
<li>Plasmid purification l3
−
</p>
+
</li>
 +
</ol>
 
<p>
 
<p>
−
 
+
dCas9-GFP 24
−
    <strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>Re Cut for frag1-3 (gib)</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>McyI, McyB</strong>
+
Re cut for Fragment1-3 (gibson assembly)
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
McyI, Mcyb
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>14 August</strong>
+
14 August  
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
−
<p>
+
<ol>
  
−
        <strong>1) Run Gel for X gib Frag 1</strong>
+
<li>Run gel for xgib Fragment 1  
−
</p>
+
</li>
 +
</ol>
 
<p>
 
<p>
−
 
+
-no bands at all  
−
    <strong>-No bands at all</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
Possibly due to pipetting errors.  
−
    <strong>Possibly due to pipetting errors.</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
−
    <strong> </strong>
+
 
</p>
 
</p>
−
<p>
+
<ol>
  
−
        <strong>2) Started 10uL reaction using Gibson concentration according to following gel. (pic Gib4)</strong>
+
<li>Started 10ul reaction using gibson concentration according to following gel. (pic gib4)  
−
</p>
+
</li>
 +
</ol>
 
<p>
 
<p>
−
 
+
−
    <strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
Reaction: Fragment3: 50ng  
−
    <strong>Reaction: Frag3: 50ng</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
    Fragment2: 38.73ng  
−
    <strong>            Frag2: 38.73ng</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
    Fragment1: 29.26ng  
−
    <strong>            Frag1: 29.26ng</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
                nebuilder: 5ul
−
    <strong>        Nebuilder: 5uL</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
h2o
−
    <strong>            H2O</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
−
    <strong> </strong>
+
 
</p>
 
</p>
−
<p>
+
<ol>
  
−
        <strong>3) Set up PCR reaction for XGib Frag2</strong>
+
<li>Set up PCR reaction for xgib Fragment2
−
</p>
+
</li>
 +
</ol>
 
<p>
 
<p>
−
 
+
Xgib Fragment3
−
    <strong>XGib Frag3</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
Line 591: Line 610:
 
<table>
 
<table>
 
   <tr>
 
   <tr>
−
   <td>98°C
+
   <td>98c
 
   </td>
 
   </td>
−
   <td>98°C
+
   <td>98c
 
   </td>
 
   </td>
−
   <td>66°
+
   <td>66c
 
   </td>
 
   </td>
−
   <td>72°C
+
   <td>72c
 
   </td>
 
   </td>
−
   <td>72°C
+
   <td>72c
 
   </td>
 
   </td>
−
   <td>4°C
+
   <td>4c
 
   </td>
 
   </td>
 
   </tr>
 
   </tr>
 
   <tr>
 
   <tr>
−
   <td>30sec
+
   <td>30sec  
 
   </td>
 
   </td>
−
   <td>5s
+
   <td>5s  
 
   </td>
 
   </td>
−
   <td>15s
+
   <td>15s  
 
   </td>
 
   </td>
−
   <td>1.5min
+
   <td>1.5min  
 
   </td>
 
   </td>
−
   <td>2min
+
   <td>2min  
 
   </td>
 
   </td>
−
   <td>¥
+
   <td>  
 
   </td>
 
   </td>
 
   </tr>
 
   </tr>
Line 622: Line 641:
  
 
<p>
 
<p>
−
 
+
−
    <strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
−
    <strong> </strong>
+
 
</p>
 
</p>
−
<p>
+
<ol>
  
−
        <strong>4) Transformed Gib 4.1 Gib 4.2</strong>
+
<li>Transformed gib 4.1 gib 4.2  
−
</p>
+
</li>
 +
</ol>
 
<p>
 
<p>
−
 
+
Overnight ligation sgrna McyI petblue
−
    <strong>Overnight ligation sgRNA mcyI Petblue</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
Sgrna Mcyb PETblue
−
    <strong>sgRNA mcyB petblue</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
gib -0.9ul dna
−
    <strong> Gib -0.9uL DNA </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
    sgrna -2ul
−
    <strong> sgRNA -2uL</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
−
    <strong> </strong>
+
 
</p>
 
</p>
−
<p>
+
<ol>
  
−
        <strong>5) Checked plates for previous transformation: Gib 3 in 1 -</strong>
+
<li>Checked plates for previous transformation: gib 3 in 1 -  
−
</p>
+
</li>
 +
</ol>
 
<p>
 
<p>
−
 
+
  gib 2     - no colonies  
−
    <strong>                                                                          Gib 2   - No colonies</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
  gib 3     - from last year -PETblue stock -yes colonies
−
    <strong>                                                                          Gib 3       -</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
−
    <strong>  From last yr -petblue stock -yes colonies</strong>
+
 
</p>
 
</p>
−
<p>
+
<ol>
  
−
    <strong> </strong>
+
<li>Picked 6 colonies from petblue
−
</p>
+
</li>
 +
</ol>
 
<p>
 
<p>
−
 
+
petblue 1-3 -3ml(with amp)
−
        <strong>6) Picked 6 colonies from petblue</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
petblue 4-5 -2ml       
−
    <strong>              Petblue 1-3 -3mL    (with</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
Master plate -no xgal iptg
−
    <strong>              Petblue 4-5 -2mL  amp)</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
−
    <strong>Master plate -No xgal Iptg</strong>
+
 
</p>
 
</p>
−
<p>
+
<ol>
  
−
    <strong> </strong>
+
<li>Run gel for PCR products 
−
</p>
+
</li>
 +
</ol>
 
<p>
 
<p>
−
 
+
Fragment 2, Fragment 3 x gib
−
        <strong>7) Run gel for pcr products</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
−
    <strong>Frag 2, frag 3 X Gib</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
Gel purification
−
    <strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
Dna concentration on 13/8
−
    <strong>Gel purification</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
−
    <strong>DNA concentration on 13/8</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
−
    <strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
−
    <strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
−
    <strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
15 August 
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>15 August</strong>
+
 
</p>
 
</p>
−
<p>
+
<ol>
−
<strong> </strong>
+
−
</p>
+
−
<p>
+
  
−
        <strong>1) RE cut for frag1 frag 2 and frag 3</strong>
+
<li>Restriction endonuclease cut for Fragment1 Fragment 2 and Fragment 3  
−
</p>
+
</li>
 +
</ol>
 
<p>
 
<p>
−
 
+
Xgib ~6 hr (MluI, BssaI and NheI)
−
    <strong>X Gib ~6 hr</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>        </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>        Nanodrop before RE cut</strong>
+
nanodrop before restriction endonuclease cut  
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
−
<p>
+
<ol>
  
−
        <strong>2) Set up Gibson reaction</strong>
+
<li>Set up gibson reaction  
−
</p>
+
−
<p>
+
  
−
            <strong>a. Frag 3 50nL 16uL              -       </strong>
+
<li>Fragment 3 50ng      16ul-  
−
</p>
+
−
<p>
+
  
−
            <strong>b. Frag 2 38.73ng 2.1 uL        -</strong>
+
<li>Fragment 2 38.73ng 2.1 ul-  
−
</p>
+
−
<p>
+
  
−
            <strong>c. Frag 1 29.26ng 0.4uL        -       20uL/xh</strong>
+
<li>Fragment 1 29.26ng 0.4ul-20ul/xh  
−
</p>
+
−
<p>
+
  
−
            <strong>d. Nebuilder         10uL          -</strong>
+
<li>Nebuilder                 10ul-  
−
</p>
+
−
<p>
+
  
−
            <strong>e. H2O              5.9uL        -</strong>
+
<li>H2o                      5.9ul-  
−
</p>
+
</li>
 +
</ol>
 
<p>
 
<p>
−
 
+
−
        <strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
50c for 2hr (optimized protocol)
−
        <strong>50C for 2hr</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
−
<p>
+
<ol>
  
−
        <strong>3) Plasmid Purification of Petblue</strong>
+
<li>Plasmid purification of petblue 
−
</p>
+
</li>
 +
</ol>
 
<p>
 
<p>
−
<strong>                    Elude using 30uL</strong>
+
            elute using 30ul 
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>        Nanodrop</strong>
+
nanodrop
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
−
<p>
+
<ol>
  
−
        <strong>4) Gib 4.1 -Petblue mcyB plasmid have 1 colony each</strong>
+
<li>Gib 4.1 -petblue mcyb plasmid have 1 colony each  
−
</p>
+
</li>
 +
</ol>
 
<p>
 
<p>
−
<strong>                    -Picked & inoculated</strong>
+
-picked & inoculated  
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
−
<p>
+
<ol>
  
−
        <strong>5) PCR purified RE cuts from (nolning?)</strong>
+
<li>PCR purified restriction endonuclease cuts from (Fragment 1, 2, 3 13/8?)  
−
</p>
+
</li>
 +
</ol>
 
<p>
 
<p>
−
 
+
-ligated h3 & 3 in 1  
−
    <strong>-ligated H3 & 3 in 1</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
1+3 1:1  
−
    <strong>        1+3 1:1</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
1+2+31:1:1  
−
    <strong>        1+2+3 1:1:1</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
−
    <strong> </strong>
+
 
</p>
 
</p>
−
<p>
+
<ol>
  
−
        <strong>6) Sequencing</strong>
+
<li>Sequencing  
−
</p>
+
</li>
 +
</ol>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>16 August</strong>
+
16 August  
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
−
<p>
+
<ol>
  
−
        <strong>1) Plasmid purification of</strong>
+
<li>Plasmid purification of
−
</p>
+
</li>
 +
</ol>
 
<p>
 
<p>
−
 
+
Gib4.1 & pet blue mcyb plasmid from 1518  
−
    <strong>Gib4.1 & pET blue mcyB plasmid from 1518</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
Gib4.1 does not seem to grow  
−
    <strong>Gib4.1 does not seem to grow</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
During inoculation  
−
    <strong>During inoculation</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
Therefore, low yield.  
−
    <strong>Therefore, low yield.</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
−
    <strong> </strong>
+
 
</p>
 
</p>
−
<p>
+
<ol>
  
−
        <strong>2) Run gel for frag 1+3 & 3in1 (ligated)</strong>
+
<li>Run gel for Fragment 1+3 & 3in1 (ligated)  
−
</p>
+
</li>
 +
</ol>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
−
<p>
+
<ol>
  
−
        <strong>3) Intended to amp 1+3 using 3F + 1R and 3R + 1F</strong>
+
<li>Intended to amp 1+3 using 3f + 1r and 3r + 1f
−
</p>
+
</li>
 +
</ol>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
−
<p>
+
<ol>
  
−
        <strong>4) Intended to check Gib4 using 1+2, 2+3, 1+3 primers.</strong>
+
<li>Intended to check gib4 using 1+2, 2+3, 1+3 primers.  
−
</p>
+
</li>
 +
</ol>
 
<p>
 
<p>
−
 
+
−
    <strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
−
    <strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>(3) and (4) failed possibily due to pcr errors</strong>
+
(3) and (4) failed possibily due to PCR errors  
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>-Use 3 mins extension       65C other times and maximum</strong>
+
-use 3 mins extension 65c other times and maximum  
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
−
<p>
+
<ol>
  
−
        <strong>5) Intended to check sgRNA mcyB petblue using colony PCR</strong>
+
<li>Intended to check sgrna mcyb petblue using colony PCR  
−
</p>
+
</li>
 +
</ol>
 
<p>
 
<p>
−
 
+
(failed due to 0.7% gel, not 1.5% gel pic in chelsy’s phone  
−
    <strong>(failed due to 0.7% gel, not 1.5% gel pic in chelsy’s phone</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>19 August</strong>
+
19 August
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
−
<p>
+
<ol>
  
−
        <strong>1) Colony PCR for McyB sgRNA petblue</strong>
+
<li>Colony PCR for mcyb sgrna petblue  
−
</p>
+
</li>
 +
</ol>
 
<p>
 
<p>
−
 
+
(only 1 white colony)  
−
        <strong>(only 1 white colony)</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
Results positive: band size ~300bp  
−
        <strong>Results positive: band size ~300bp</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
−
        <strong> </strong>
+
 
</p>
 
</p>
−
<p>
+
<ol>
  
−
        <strong>2) Inoculated 2 3mL tubes of Gib4</strong>
+
<li>Inoculated 2 3ml tubes of gib4
−
</p>
+
</li>
 +
</ol>
 
<p>
 
<p>
−
 
+
-gib4.1 & 4.2  
−
        <strong>-Gib4.1 & 4.2</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
From master plate  
−
        <strong>From master plate</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
−
        <strong> </strong>
+
 
</p>
 
</p>
−
<p>
+
<ol>
  
−
        <strong>3) Set up overnight PCR to amp mcyI, frag1, frag3 xgib,</strong>
+
<li>Set up overnight PCR to amp mcyi, Fragment1, Fragment3 xgib,  
−
</p>
+
</li>
 +
</ol>
 
<p>
 
<p>
−
 
+
−
    <strong> </strong>
+
 
</p>
 
</p>
−
<p>
+
<ol>
  
−
        <strong>4) RE cut mcyI (247.8ng), petblue (1000ng), frag1 (741ng)- frag3 (140ng) overnight [McyI only added 3uL]</strong>
+
<li>Restriction endonuclease (PstI, HindIII, MluI, BssaI, NheI) cut mcyi (247.8ng), petblue (1000ng), Fragment1 (741ng)- Fragment3 (140ng) overnight [mcyi only added 3ul]  
−
</p>
+
</li>
 +
</ol>
 
<p>
 
<p>
−
 
+
−
    <strong> </strong>
+
 
</p>
 
</p>
−
<p>
+
<ol>
  
−
        <strong>5) Checked Neb 3 in 1 using different primers</strong>
+
<li>Checked neb 3 in 1 using different primers
−
</p>
+
</li>
 +
</ol>
 
<p>
 
<p>
−
 
+
Checked Fragment 1+3  
−
    <strong>Checked frag 1+3</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> 20 August</strong>
+
20 August
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
−
<p>
+
<ol>
  
−
        <strong>1) Run gel for mcyI, frag1-3 xGib</strong>
+
<li>Run gel for mcyi, Fragment1-3 xgib
−
</p>
+
</li>
 +
</ol>
 
<p>
 
<p>
−
 
+
-gel purification takara
−
    <strong>-gel purification Takara</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
−
    <strong> </strong>
+
 
</p>
 
</p>
−
<p>
+
<ol>
  
−
        <strong>2) PCR clean mcyI, petblue 2 from overnight RE</strong>
+
<li>PCR clean mcyi, petblue 2 from overnight restriction endonuclease cut
−
</p>
+
</li>
 +
</ol>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
−
<p>
+
<ol>
  
−
        <strong>3) Ligated mcyI petblue, frag1+3</strong>
+
<li>Ligated mcyi petblue, Fragment1+3  
−
</p>
+
</li>
 +
</ol>
 
<p>
 
<p>
−
 
+
-vector 100ng-vector 100ng  
−
    <strong>-vector 100ng -vector 100ng</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>        1:3                     1:1</strong>
+
1:3 1:1  
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
 +
<ol>
 +
 +
<li>?
 +
</li>
 +
</ol>
 
<p>
 
<p>
−
 
+
−
        <strong>4) ?</strong>
+
 
</p>
 
</p>
−
<p>
+
<ol>
  
−
    <strong> </strong>
+
<li>Plasmid purification gib6.4
−
</p>
+
</li>
 +
</ol>
 
<p>
 
<p>
−
 
+
-yield extremely low
−
        <strong>5) Plasmid pur Gib6.4</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
−
    <strong>-yield extremely low</strong>
+
 
</p>
 
</p>
−
<p>
+
<ol>
  
−
    <strong> </strong>
+
<li>Picked colonies: 2 nebuilder 3in1.-
−
</p>
+
</li>
 +
</ol>
 
<p>
 
<p>
−
 
+
    2 gib4  -master plate
−
        <strong>6) Picked colonies: 2 Nebuilder 3in1.            -</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>                                  2 Gib4                          -       Master Plate</strong>
+
  2 sgrna mcyb petblue-  
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>                                  2 sgRNA mcyB petblue  -</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
21 August
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>21 August</strong>
+
 
</p>
 
</p>
 +
<ol>
 +
 +
<li>Plasmid pur for 6 tubes -nebuilder x2, gib4 x2, sgrna mcyb petblue
 +
</li>
 +
</ol>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
 +
<ol>
 +
 +
<li>Collected overnight ligation
 +
</li>
 +
</ol>
 
<p>
 
<p>
−
 
+
-amplified Fragment 1+3 wing
−
        <strong>1) Plasmid pur for 6 tubes -NEbuilder x2, gib4 x2, sgRNA mcyB petblue</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
Primer Fragment1 f & primer Fragment3 r (vice versa also)
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
−
        <strong>2) Collected overnight ligation</strong>
+
 
</p>
 
</p>
−
<p>
+
<ol>
  
−
    <strong>-Amplified frag 1+3 wing</strong>
+
<li>Transformed sgrna mcyi petblue and dcas9 gfp (l3)
−
</p>
+
</li>
 +
</ol>
 
<p>
 
<p>
−
<strong>Primer frag1 F & primer frag3 R (vice versa also)</strong>
+
 
</p>
 
</p>
 +
<ol>
 +
 +
<li>Received sanger sequence results 
 +
</li>
 +
</ol>
 
<p>
 
<p>
−
<strong> </strong>
+
-99.4% identical
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
mutation- x
−
        <strong>3) Transformed sgRNA mcyI petblue and dCas9 gfp (L3)</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
−
    <strong> </strong>
+
 
</p>
 
</p>
−
<p>
+
<ol>
  
−
        <strong>4) Received sanger sequence results</strong>
+
<li>Picked 25 colonies from previous l3 plate
−
</p>
+
</li>
 +
</ol>
 
<p>
 
<p>
−
 
+
-colony PCR 25-49  -one taq
−
    <strong>-99.4% identical</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
50- sapphire
−
    <strong>        Mutation- X</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
−
<p>
+
<ol>
  
−
        <strong>5) Picked 25 colonies from previous L3 plate</strong>
+
<li>Single cut & double cut 3 in 1 plasmid
−
</p>
+
</li>
 +
</ol>
 
<p>
 
<p>
−
 
+
-all undesired results
−
    <strong>-colony PCR          25-49  -one Taq</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
−
    <strong>                                50    - sapphire</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
22 Aug
−
    <strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
gibson assembly ,incubate 2h
−
        <strong>6) Single cut & double cut 3 in 1 plasmid</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
run gel for colony PCR and Fragment 1+3 and the overnight cut of 3 in 1
−
    <strong>-All undesired results</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
For colonies with correct band after colony PCR, pick colony from master plate, inoculate
−
    <strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> 22aug</strong>
+
Fragment 1+3, Fragment 2 re cut, 
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> Gibson assembly ,incubate 2h</strong>
+
Send mcyb to sequencing
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> Run gel for colony pcr and frag 1+3 and the overnight cut of 3 in 1</strong>
+
Pick colony from plates  if have
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>For colonies with correct band after colony pcr, pick colony from master plate, inoculate</strong>
+
Transform gibson products
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>Frag 1+3, frag 2 re cut,</strong>
+
Ligate Fragment 2 to 1+3  
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>Send mcyB to sequencing</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>pick colony from plates if have</strong>
+
   
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>Transform gibson products</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>Ligate frag 2 to 1+3</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
23 Aug
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
Run gel for PCR dcas9 gfp, 
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
Run gel for PCR 1+3 sgrna mcyi pet blue
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>23 aug</strong>
+
26 Aug
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>Run gel for Pcr dcas9 gfp,</strong>
+
Run gel for previous 40 colony PCR
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>Run gel for Pcr 1+3 sgrna mcyI pet blue</strong>
+
Inoculate l3 with correct band size
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
Preparation for electroporation
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>26 aug</strong>
+
Transform ligation product and  3 in 1 for characterization
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>Run gel for previous 40 colony pcr</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>Inoculate L3 with correct band size</strong>
+
27 Aug
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>Preparation for electroporation</strong>
+
Pick clone for previous transformation
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>Transform ligation product and  3 in 1 for characterization</strong>
+
3 in 1 plasmid pur using traditional method
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
Restriction endonuclease cut gib 5 (MluI)
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>27 aug</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>Pick clone for previous transformation</strong>
+
28 Aug
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>3 in 1 plasmid pur using traditional method</strong>
+
PCR screening mcyi, b
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>Re cut gib 5</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
29 Aug
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>28 aug</strong>
+
Plasmid purification. Using tradition n takara
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>PCR screening mcyI, B</strong>
+
Inoculate gib 5, prepare for measurement
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>29 aug</strong>
+
30 Aug 
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>Plasmid pur. Using tradition n takara</strong>
+
I think no lab
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>Inoculate gib 5, prepare for measurement</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
3/9
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>30 aug</strong>
+
Restriction endonuclease cut dcas9 gfp (EcoRI, PstI)
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>I think no lab</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
4/9
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>31 aug</strong>
+
Run gel for restriction endonuclease cut dcas9 gfp
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>Pick sgRNA clone?</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
5/9
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>1/9</strong>
+
Transformation inoculate pet blue, puc19, 3 in 1, psb1c3 transformants
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>I think no lab</strong>
+
Plasmid purification 3 in 1 using takara plasmid purification kit
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>2/9</strong>
+
6/9  
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>I think no lab</strong>
+
Plasmid purification 3 in 1 using takara plasmid purification kit
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>3/9</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>Re cut dcas9 gfp</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
9/9
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>4/9</strong>
+
2 restriction endonuclease cuts (dCas9-GFP-sgRNA)
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>Run gel for re cut dcas9 gfp</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
10/9
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>5/9</strong>
+
Run gel for the colony PCR products (1.5 gel, all in) 65v 45 min 700 or no band
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>Transformation inoculate pet blue, puc19, 3 in 1, psb1c3 transformants</strong>
+
Pick colony and colony PCR 
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>Plasmid purification 3 in 1 using Takara plasmid purification kit</strong>
+
Pour agar plate 
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
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>
−
<strong>6/9</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>Plasmid purification 3 in 1 using takara plasmid purification kit</strong>
+
11/9
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
PCR clean for 3 in 1 cut
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
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>
 
<p>
 
<p>
−
<strong> </strong>
+
Gel purification for the above cut gel (gm buffer 650ul)
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>9/9</strong>
+
Run gel for colony PCR yesterday (1.5 gel)
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>2 re cuts</strong>
+
Set ligation mixture (restriction endonuclease cuts of  3 in 1 and mcy dcas9 gfp) 1:1
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>10/9</strong>
+
12/9  
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>run gel for the colony pcr products (1.5 gel, all in) 65V 45 min 700 or no band</strong>
+
Heat inactivate ligation
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>Pick colony and colony pcr</strong>
+
2. Transform ligation (total dna 60ng)
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>pour agar plate</strong>
+
3. Dilute overnight inoculation, 1 to 3
 
</p>
 
</p>
 
<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>
+
4. Set restriction endonuclease cut of 3 in 1 and sgrna dcas9 gfp
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>11/9</strong>
+
13/9  
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>PCR clean for 3 in 1 cut</strong>
+
Measure od (growth curve) for modelling
 
</p>
 
</p>
 
<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>
 
<p>
 
<p>
−
<strong>Gel pur for the above cut gel (GM buffer 650ul)</strong>
+
16/9
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>Run gel for colony pcr yesterday (1.5 gel)</strong>
+
Restriction endonuclease cuts for final product (XhoI, XbaI)  
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>Set ligation mixture (re cuts of  3 in 1 and Mcy dcas9 gfp) 1:1</strong>
+
Pick colony for growth curve
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>12/9</strong>
+
17/9  
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>Heat inactivate ligation</strong>
+
Growth curve
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>2. Transform ligation (total dna 60ng)</strong>
+
Run gel for the restriction endonuclease cuts, PCR clean, start ligation  
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>3. Dilute overnight inoculation, 1 to 3</strong>
+
Transform ligation mixture
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>4. Set RE of 3 in 1 and sgrna dcas9 gfp</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
18/9
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>13/9</strong>
+
Transofrmation of final ligation products
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>Measure OD (growth curve) for modelling</strong>
+
2. Od measurements
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>                                          </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>16/9</strong>
+
19/9  
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>Re cuts for final product</strong>
+
pick colony (fat clone)
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>pick colony for growth curve</strong>
+
Transformation final product
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>17/9</strong>
+
20/9  
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>growth curve</strong>
+
Pick clone
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>Run gel for the RE cuts, pcr clean, start ligation</strong>
+
PCR check band 
 
</p>
 
</p>
 +
<ul>
 +
 +
<li>Correct 
 +
</li>
 +
</ul>
 
<p>
 
<p>
−
<strong>transform ligation mixture</strong>
+
Plasmid purification by PETER failed cuz me flip his gel…
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>18/9</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>Transofrmation of final ligation products</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>2. OD measurements</strong>
+
23/9
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
Pick colony of final product
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>19/9</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> Pick colony (fat Clone)</strong>
+
24/9
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>Transformation final product</strong>
+
Plasmid purification
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>20/9</strong>
+
25/9
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>Pick clone</strong>
+
Nanodrop final product
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>Pcr check band</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
26/9
−
        <strong>·  Correct </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>Plasmid pur by wch failed cuz me filp his gel..</strong>
+
Restriction endonuclease cut for final product (PstI)
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
27/9
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
Run gel for the restriction endonuclease cuts and final product 24 and 15. 
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>23/9</strong>
+
All in for restriction endonuclease cuts, 
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>pick colony of final product</strong>
+
5ul for final product 24 and 15
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
65v 50min
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>24/9</strong>
+
Nanodrop final product
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>25/9</strong>
+
30/9  
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>Nanodrop final product</strong>
+
Transformation
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
final product plasmid 15 17 24 29(bl21)
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>26/9</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>27/9</strong>
+
3/10
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>run gel for the RE cuts and final product 24 and 15.</strong>
+
Observe green fluorescence by uv exposure
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>All in for re cuts,</strong>
+
Inoculate
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>5ul for final product 24 and 15</strong>
+
Observe green fluorescence again by uv exposure
 
</p>
 
</p>
 +
<ul>
 +
 +
<li>All negative 
 +
</li>
 +
</ul>
 
<p>
 
<p>
−
<strong>65V 50min</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>Nanodrop final product</strong>
+
4/10
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
Plasmid purification of final product
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>30/9</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>transformation</strong>
+
7/10
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> final product plasmid 15 17 24 29(BL21)</strong>
+
Transformation of final products into DH5a
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>1/10</strong>
+
8/10  
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
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>
−
<strong>2/10</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
9/10
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>3/10</strong>
+
Filtered Vitamin B12
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>observe green fluorescence by uv exposure</strong>
+
Autoclave bold3n
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>inoculate</strong>
+
Add to agar
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>observe green fluorescence again by uv exposure</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
 
−
        <strong>·  All negative</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>4/10</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>7/10</strong>
+
10/10
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
Plasmid purification with ethanol
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>8/10</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>Prepared 0.1mM NaCl, 0.1mM HEPES, 0.1mM Vitb12, all not sterilized. Picked 10 colonies, inoculated in 20mL Lb</strong>
+
11/10
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
Measure Optical density (growth curve)
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>10/10</strong>
+
Transformation of microcystis
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>Plasmid purification with ethanol</strong>
+
Electroporation
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>Filter b12</strong>
+
Ligation by Peter
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
Dna purification 
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>11/10</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>OD (growth curve)</strong>
+
14/10
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>Transformation of microcystis</strong>
+
Transformation of previous ligation
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>Electroporation</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>Ligation by wch</strong>
+
15/10
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>Dna purification</strong>
+
Pick clones, inoculate i12
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
Standard solution
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>16/10</strong>
+
16/10  
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>Pour plate with kanamycin resistance and chl </strong>
+
Pour plate with kanamycin resistance and chloramphenicol 
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>Plasmid Purification of Insert 1 and Insert 2</strong>
+
Takara plasmid purification of insert 1 and insert 2  
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>18/10</strong>
+
17/10  
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>Pick Insert 1 and 2 ,BL21 and NEB5a clones</strong>
+
Peter ethanol precipitation of final product
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
Electroporation (natural tranformation protocol)
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong>Missing</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
Ligation mixture (cut with SpelI, xbaI)
−
        <strong>1.    20-21/7</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
Run gel for plasmid purifiction product of i12
−
        <strong>2.    23/7-8/8</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
Nanodrop i12
−
        <strong>3.    24/9 (no gel pic n whatsapp)</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
Anson adjust pH for bg11
−
        <strong>4.    26/9 (no gel pic n whatsapp)</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
Error: labelling error for plasmid pu
−
        <strong>5.    1/10 (no gel pic n whatsapp)</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
−
        <strong>6.    2/10(no gel pic n whatsapp)</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
18/10  
−
        <strong>7.    4/10(no gel pic n whatsapp)</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
 
+
Pick insert 1 and 2 ,BL21 and NEB5a clones 
−
        <strong>8.    7/10(no gel pic n whatsapp)</strong>
+
 
</p>
 
</p>
 
<p>
 
<p>
−
<strong> </strong>
+
 
</p>
 
</p>
−
+
</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