- Inoculate 2 x 500 ml of LB with 1/100 volume of a fresh overnight culture.
- Grow cells at 37°C with vigorous shaking to an OD600 of 1.0.
- Transfer the cultures to an ice/water bath and keep or 10 min.
- Centrifuge at 7000 g for 20 min at 4 °
- Resuspend pellets in a total of 2 x 250 ml ice-cold MQ water. Centrifuge.
- Resuspend pellets in a total of 2 x 100 ml ice-cold MQ water. Centrifuge.
- Resuspend in 20 ml 10% glycerol. Tranfser to a small centrifuge tube. Centrifuge.
- Resuspend in a small final volume of 10% glycerol.
- Freeze at - 80°C the suspension in aliquots of 40 μL.
- Thaw the cells on ice for 10 minutes.
- Add 1-2 μL of DNA. Mix and keep on ice for 1 minute.
- Set the Gene Pulser apparatus at 25 μF and 2.5 kV. Set the pulse Controller to 400W.
- Transfer the mixture of cells and DNA to a cold 0.2 cm electroporation cuvette. Place the cuvette in a chilled safety chamber slide, push the slide into the chamber until the cuvette is seated between the contacts in the base of the chamber.
- Pulse once at the above settings. (Time constant should be 4.5 – 5 msec. Field strength will be 12.5 kV/cm).
- Remove the cuvette from the chamber and immediately add 1 ml cold SOC medium to the cuvette. Mix and transfer to a 10 ml test tube. Incubate at 37°C for 1 hour.
- Plate on selective media.
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