Fong:Electrocompetency: Difference between revisions
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#Inoculate 500mL of fresh LB with a 5mL overnight culture | #Inoculate 500mL of fresh LB with a 5mL overnight culture | ||
#Shake at 37C and grow until OD600=0.6-1.0 | #Shake at 37C and grow until OD600=0.6-1.0 | ||
# | #Split cells into two chilled, sterile centrifuge bottles - chill for 20 minutes | ||
# | #Spin at 3000rpm for 15 minutes at ~0C and discard all of the supernatant | ||
#Gently resuspend cells in 25mL of ice-cold 10% glycerol | |||
#Spin at 3000rpm for 15 minutes at ~0C and discard all of the supernatant | |||
#Gently resuspend cells in 15mL of ice-cold 10% glycerol | |||
#Transfer cells to two chilled, sterile 15mL conical tubes | |||
#Spin at 3000rpm for 15 minutes at ~0C and discard all of the supernatant | |||
#Gently resuspend cells in 2.5mL of ice-cold 10% glycerol | |||
Revision as of 13:46, 3 June 2009
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Protocol for the preparation of electrocompetent E. coli stocks
Materials
Procedure
- Inoculate 500mL of fresh LB with a 5mL overnight culture
- Shake at 37C and grow until OD600=0.6-1.0
- Split cells into two chilled, sterile centrifuge bottles - chill for 20 minutes
- Spin at 3000rpm for 15 minutes at ~0C and discard all of the supernatant
- Gently resuspend cells in 25mL of ice-cold 10% glycerol
- Spin at 3000rpm for 15 minutes at ~0C and discard all of the supernatant
- Gently resuspend cells in 15mL of ice-cold 10% glycerol
- Transfer cells to two chilled, sterile 15mL conical tubes
- Spin at 3000rpm for 15 minutes at ~0C and discard all of the supernatant
- Gently resuspend cells in 2.5mL of ice-cold 10% glycerol
