Team:Paris Liliane Bettencourt/Notebook/2011/08/05/
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(Created page with "== Cyrille == The bands extracted yesterday will be completed and digested using Taq ploymerase. We will use the the Taq polymerase mastermix. The protocol will be: 25µL of F...") |
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10µL of the final product is then transformed by heat chock with 100µL of MH1 competent cells | 10µL of the final product is then transformed by heat chock with 100µL of MH1 competent cells | ||
+ | |||
+ | == Kevin == | ||
+ | |||
+ | === Gel verification of Dave Lane plasmids === | ||
+ | In a 1% agar gel, we have test our plasmids digested by XbaI and PstI enzyme. We've look if lines that we should see are the good one. | ||
+ | |||
+ | [[File:gelverifdlplasmid.jpg|450px|thumb|center|Gel of verification Dave Lane plasmids digestion]] | ||
+ | |||
+ | *Column 1 : expected 1075/1,077/4236 pb | ||
+ | *Column 2 : expected 520/838/4520 pb | ||
+ | *Column 3 : expected 450/833/950/1545/2110 pb | ||
+ | *Column 4 : expected 402/833/950/1545/2110 pb |
Revision as of 17:36, 5 August 2011
Cyrille
The bands extracted yesterday will be completed and digested using Taq ploymerase. We will use the the Taq polymerase mastermix.
The protocol will be:
25µL of Fermentas PCR mastermix (2x) 25µL DNA template
The PCR cycles will be:
- 80°C 1 min
- 80°C 30 sec
- 55°C 30 sec
- 72°C 30 sec
-> 10 cycles
- 72°C 5 min
- 4°C forever
The PCR product is treated with the PCR product purification kit. The tubes contains 20.1, 21, and 30 ng/µL resp.
Then the final product is ligated using T4 DNA ligate.
The mix contains: - Linear DNA : 2µL (40-50 ng final) - 10 T4 DNA ligase buffer: 5µL - 50% PEG 4000 solution: 5µL - T4 DNA ligase: 1µL - DNAse free water: 37 µL
Then it is incubated for 1h at 22°C.
10µL of the final product is then transformed by heat chock with 100µL of MH1 competent cells
Kevin
Gel verification of Dave Lane plasmids
In a 1% agar gel, we have test our plasmids digested by XbaI and PstI enzyme. We've look if lines that we should see are the good one.
- Column 1 : expected 1075/1,077/4236 pb
- Column 2 : expected 520/838/4520 pb
- Column 3 : expected 450/833/950/1545/2110 pb
- Column 4 : expected 402/833/950/1545/2110 pb