Zashaver: Created page with " == Electroporation of JM109 or Similar Strain == '''Preparing Electrocompetent ''E. coli''''' (note: all glassware and solutions must be sterile) # Inoculate a 50 mL flask o..."

2016-06-23T21:55:51Z

Created page with " == Electroporation of JM109 or Similar Strain == '''Preparing Electrocompetent ''E. coli''''' (note: all glassware and solutions must be sterile) # Inoculate a 50 mL flask o..."

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== 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 in this solution, it may arc and the cuvette may explode. 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.

Electroporation Old School Campbell - Revision history

Zashaver: Created page with " == Electroporation of JM109 or Similar Strain == '''Preparing Electrocompetent ''E. coli''''' (note: all glassware and solutions must be sterile) # Inoculate a 50 mL flask o..."