Team:Paris Bettencourt/Notebook/Drug Screening/Monday 19th August.html

From 2013.igem.org

Drug Screening

Day 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.