实验步骤

1. Thaw tubes of competent cells on ice, occasionally inverting gently to mix.

2. Pre-cool Falcon 2059 tubes on ice.

3. Aliquot 30 ul cells into each tube.

4. Add up to 10 ul DNA in TE or ligation buffer and place on ice for 10 minutes. This allows DNA to attatch to cells.

5. Place in 42 degree water bath for 1 minute. This treatment shocks the cell membranes to allow take-up of the DNA.

6. Add 0.5 ml of L2 media and shake at 37 degrees for up to 1-2 hours. Remember: Do not use media containing selection agent, usually ampicillin, as this is the period in which antibiotic resistance develops.

7. Pipet cells onto L2 plates containing selective agent and spread using a sterile glass spreader (a bent Pasteur pipet).

8. Wait until all liquid has been absorbed into plate, then incubate overnight at 37 degrees.