Fong:Electrocompetency: Difference between revisions
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==Protocol for the preparation of electrocompetent ''E. coli'' stocks== | ==Protocol for the preparation of electrocompetent ''E. coli'' stocks== | ||
==Materials== | ==Materials== | ||
*Overnight culture of particular strain (e.g., DH5α, NEB10β, DB3.1) | |||
*Bucket of ice | |||
*Refrigerated centrifuge | |||
*Ice-cold 10% glycerol | |||
*Chilled, sterile centrifuge bottles | |||
*Chilled, sterile 15mL conical tubes | |||
*Chilled, sterile cryogenic vials | |||
==Procedure== | ==Procedure== | ||
#Inoculate 500mL of fresh LB with a 5mL overnight culture | #Inoculate 500mL of fresh LB with a 5mL overnight culture | ||
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#Spin at 3000rpm for 10 minutes at ~0C and discard all of the supernatant | #Spin at 3000rpm for 10 minutes at ~0C and discard all of the supernatant | ||
#Gently resuspend cells in 2.5mL of ice-cold 10% glycerol | #Gently resuspend cells in 2.5mL of ice-cold 10% glycerol | ||
#Split cell suspension into 10 chilled, sterile | #Split cell suspension into 10 chilled, sterile cryogenic vials (250μL aliquots) | ||
#Label and store vials in -80C freezer | |||
==Notes== | ==Notes== | ||
*This stock will be viable for at least 6 months | *This stock will be viable for at least 6 months | ||
*Do not re-freeze aliquots once they have thawed | *Do not re-freeze aliquots once they have thawed |
Latest revision as of 13:55, 3 June 2009
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Protocol for the preparation of electrocompetent E. coli stocks
Materials
- Overnight culture of particular strain (e.g., DH5α, NEB10β, DB3.1)
- Bucket of ice
- Refrigerated centrifuge
- Ice-cold 10% glycerol
- Chilled, sterile centrifuge bottles
- Chilled, sterile 15mL conical tubes
- Chilled, sterile cryogenic vials
Procedure
- Inoculate 500mL of fresh LB with a 5mL overnight culture
- Shake at 37C and grow until OD600=0.6-1.0
- Split cell suspension into two chilled, sterile centrifuge bottles - chill for 20 minutes
- Spin at 3000rpm for 20 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 10 minutes at ~0C and discard all of the supernatant
- Gently resuspend cells in 2.5mL of ice-cold 10% glycerol
- Split cell suspension into 10 chilled, sterile cryogenic vials (250μL aliquots)
- Label and store vials in -80C freezer
Notes
- This stock will be viable for at least 6 months
- Do not re-freeze aliquots once they have thawed