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Team:Bordeaux/NotebookAugust22
From 2014.igem.org
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RFP (706pb) 12,8ng/μl<br></div><br> | RFP (706pb) 12,8ng/μl<br></div><br> | ||
| - | We want to use 25 fmol | + | We want to use 25 fmol => we use promega website (μg to pmol converter)<br> |
<div style="margin-left:25px;"> | <div style="margin-left:25px;"> | ||
| - | ELP = 0,002pmol/μl | + | ELP = 0,002pmol/μl => 25 fmol = 12,5μl<br> |
| - | RFP = 0,027pmol/μl | + | RFP = 0,027pmol/μl => 25 fmol = 0,9μl<br><br> |
Do the mix on ice :<br> | Do the mix on ice :<br> | ||
| Line 41: | Line 41: | ||
Dilution SLP 3,6μl +6,4μl H2O<br> | Dilution SLP 3,6μl +6,4μl H2O<br> | ||
Dilution RLP 1,97μl +8,03μl H2O<br> | Dilution RLP 1,97μl +8,03μl H2O<br> | ||
| - | </div> | + | </div><br> |
incubation 1h30 at 37°C :<br> | incubation 1h30 at 37°C :<br> | ||
| Line 54: | Line 54: | ||
- Enzyme inactivated 20min at 80°C <br> | - Enzyme inactivated 20min at 80°C <br> | ||
- Gel 1% (30min, 100V) <br> | - Gel 1% (30min, 100V) <br> | ||
| - | + | <br> | |
<li>Ligation</li> | <li>Ligation</li> | ||
<table> | <table> | ||
| Line 80: | Line 80: | ||
=> 30min at 16°C | => 30min at 16°C | ||
| - | + | <br><br> | |
| - | <li>Transformation</li><ul> | + | <li>Transformation</li></ul> |
</li> | </li> | ||
</ul> | </ul> | ||
Latest revision as of 02:16, 18 October 2014
22nd August
Molecular biology
-
Chewback : purification with Qiagen Kit, dosage on nanodrop
ELP (6240pb) 8,2 ng/μl
RFP (706pb) 12,8ng/μl
We want to use 25 fmol => we use promega website (μg to pmol converter)
ELP = 0,002pmol/μl => 25 fmol = 12,5μl
RFP = 0,027pmol/μl => 25 fmol = 0,9μl
Do the mix on ice :
RFP 0,9μl Plasmid 12,5μl Master Mix 12,5μl
Inoculate immediately 1h at 50°C
- Transformation of competent cells and spread on LB+Amp plates
- Gel 1% (30min, 100V)
-
Biobrick assembly
- Digestion
- Ligation
- Transformation
Dilution ELP 2,1μl +7,7μl H2O
Dilution SLP 3,6μl +6,4μl H2O
Dilution RLP 1,97μl +8,03μl H2O
incubation 1h30 at 37°C :
- Enzyme inactivated 20min at 80°CEnzyme 1 (Nhe1 or X) 0,5μl Enzyme 2 (S) 0,5lμl 10X Buffer 4 5μl DNA 10μl H2O qsp 50μl
- Gel 1% (30min, 100V)
=> 30min at 16°CPlasmid 4μl Insert 4μl Buffer 10X 1μl Taq ligase 0,5μl H2O qsp 10μl
Purification
- We change the water of dialysis at 9h.
