Electrocompetent E. coli cells
Competent cells should never be vortexed, as this will cause them to lyse and release salts into the media. Resuspend cells by pipetting up and down with a large Pasteur pipet. Once they are chilled, cells should be continuously cooled.
- prepare ON culture (5 mL of LB Amp)
- dilute the cells 100x in 100 mL LB Amp
- grow at 30 °C for about 90 min
- harvest the cells when they reach an OD600 of between 0.6 and 0.8
- split the culture into 2 x 50 mL falcon tubes, on ice
- centrifuge (10 min, 4000 rpm, 4 °C)
- wash and combine the cells
- discard the supernatant
- resuspend the cells in 2 x 25 mL of ice cold water
- combine the volumes in a single 50 mL falcon tube
- wash the cells 2 more times
- centrifuge (10 min, 4000 rpm, 4 °C)
- resuspend in 50 mL of ice cold water
- repeat centrifugation and resuspension
- wash and concentrate the cells for electroporation
- centrifuge (10 min, 4000 rpm, 4 °C)
- resuspend in 1-2 mL of ice cold water
- Use 200 µL of washed cells per transformation.
