Electroporation - Campbell Old School Method
Electroporation of JM109 or Similar Strain
Preparing Electrocompetent E. coli (note: all glassware and solutions must be sterile)
- Inoculate a 50 mL flask of LB (low salt) with JM109 stock and grow overnight at 37˚C.
- First thing in the morning, put 5 mL of overnight JM109 into a 1 liter flask containing 500 mL of LB (low salt) and incubate at 37˚C and 400 rpm.
- Monitor growth and stop when cell density is between OD600 0.5 and 1.0.
- Centrifuge cells for 15 minutes at 4˚C and 4000g (5.1K rpm in Beckman rotor JA-14) in 250 mL bottles.
- Resuspend cells in 1 liter of distilled H20; make sure there are no clumps of cells. Pellet cells again.
- Resuspend cells in 500 mL fo distilled H20; make sure there are no clumps of cells. Pellet cells again.
- Resuspend cells in 250 mL of distilled H20; make sure there are no clumps of cells. Pellet cells again.
- Resuspend cells in 20 mL of distilled H20 and transfer to two 30 mL corex tubes; make sure there are no clumps of cells. Pellet cells again for 15 minutes at 4˚C and 4000g (5.7K rpm in Beckman rotor JA-20).
- Resuspend cells in 2.5 mL of 10% glycerol - filter sterilized; make sure there are no clumps of cells.
- Aliquot cells in 50 uL volumes into sterile 500 uL microfuge tubes and store at -70˚C.
- Thaw cells on ice for at least 7 minutes.
- Set the voltage for 1.3 kilovolts.
- Place disposable cuvette with 1 mm gap on ice for at least one minute.
- In the microfuge tube containing the comp. cells, mix the DNA to be transformed with the electrocompetent cells.
- Transfer cells and DNA into disposable cuvette using the sterile dropper provided. Flick the cuvette so that cells touch all four walls of the cuvette.
- Quickly dry the outside of the cuvette and put it into the electrode chamber.
WARNINGIf there is too much salt, the cuvette can arch and make a pop sound. see example Protect yourself appropriately.
- Press the red charge/pulse button. When it beeps, the pulse has fired.
- Very quickly, add 960 uL of fresh LB medium to the cells, mix the cells, transfer the cells to sterile 12 X 75 mm plastic tubes, and incubate at 37˚C at 225 rpm for 1 hour.
- Plate the cells on the appropriate agar plates and spread the cells properly.
- Incubate overnight at 37˚C.