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From 2010.igem.org

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Revision as of 12:46, 5 July 2010

Transformation

Materials:

- competent cells

- LB medium (warmed to room temperature)

- DNA ligation mix

- LB plates containing 15-100 μg/mL antibiotic of choice, pre-warmed to 37 °C

- Water bath at 37 °C

- Shaking incubator at 37 °C.

Protocol:

1. Add 50-100 ng DNA into a 30 μL competent E.coli, and mix gently. Do not mix by pipetting up and down!

2. Incubate tube vial on ice for 30 minutes.

3. Heat-shocks the cells for exactly 5 minutes at 37 °C without shaking.

4. Immediately transfer the tubes back to ice for 2 minutes.

5. Add 250 μL of room temperature LB medium.

6. Cap tube tightly and shake tube horizontally (225 rpm) at 37 °C for 1 hour.

7. Plate from each tube 100 μL on an agar plate containing antibiotic. Spin tube, discard supernatant to leave no more than 100 μL, vortex and plate on an agar plate.

8. Incubate plates overnight at 37 °C.