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Team:Paris Bettencourt/Notebook/Drug Screening/Monday 19th August.html
From 2013.igem.org
Drug Screening
ASDFDay Numbersuffix Month
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Received FrpA and FdxA from IDT.Started cloning strategy:
Ligated FdxA G block into pJET plasmid for stock purposes, using the following protocol:
1. Set up the blunting reaction on ice:
|
Component |
Volume |
|
2X Reaction Buffer |
10 µl |
|
Non-purified PCR product or purified PCR product/other sticky-end DNA fragment |
1 µl 0.15 pmol ends |
|
Water, nuclease-free |
to 17 µl |
|
DNA Blunting Enzyme |
1 µl |
|
Total volume |
18 µl |
Vortex briefly and centrifuge for 3-5 s.
2. Incubate the mixture at 70°C for 5 min. Chill on ice.
3. Set up the ligation reaction on ice. Add the following to the blunting reaction mixture:
|
Component |
Volume |
|
pJET1.2/blunt Cloning Vector (50 ng/µl) |
1 µl (0.05 pmol ends) |
|
T4 DNA Ligase |
1 µl |
|
Total volume |
20 µl |
Vortex briefly and centrifuge for 3-5 s to collect drops.
4. Incubate the ligation mixture at room temperature (22°C) for 5 min.
