View source for 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.

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'''Electroporation Procedure'''
# 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.

'''WARNING'''If there is too much salt, the cuvette can arch and make a pop sound. [https://vimeo.com/user37250198/review/172478413/c3537bdf0e 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.