Team:Paris Liliane Bettencourt/Notebook/2011/08/05/

From 2011.igem.org

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.

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

Danyel & Camille

10 µL of each tube was stocked separately. The rest of the PCR products were digested with DpnI. The non digested mixes and the digested mixes were migrated on gel.

Non digested mixes were migrated on the first four columns. The non digested mixes were migrated on the four columns on the right separated from the non digested mixes with a blank column. The order of the digested columns seem to have been reversed.