by Wen Dong, Nov 2005

1. Prepare LB agar plates containing 100 ug/ml ampicillin.

2. Add 10 pg (1 ul) plasmid DNA (any, but with ampicillin resistance marker; the marker may be against other antibiotics such as kanamycin but then you mustr plate on kanamycin-agar plates) into 50 ul of thawed competent cells. Gently mix with pipette tip and incubate on ice for 30 min.

3. Transfer to a 42 deg water bath and hold for 30-90 sec (depending on the type of tube being used, the source of the cells, etc.).

4. Move to ice to chill for 1-2 min.

5. Add 250 ul SOC medium, warm to 37 deg in a water bath, and then move to a shaking incubator (less than 225 rpm) at 37 deg for 45 min.

6. Plate 25-300 ul on the one or many agar plates and incubate at 37 deg overnight.

7. Calculate the transformation efficiency as transformants per ug of plasmid DNA.

For chemically competent cells, use the formula below to calculate transformation efficiency:

(no. of colonies / 10 pg transformed DNA) x (10⁶ pg / ug) x (300 ul total transformation volume/ X ul plated) = (# transformants / ug plasmid DNA)

Also see these methods to prepare competent cells - this and this