Team:York/Protocols/CompetentCells
Competent Cells
Materials
- LB media (in 50 mL in 250 conical flask) pre- autoclaved (x2)
- overnight culture of DH5α (x2)
- shaker in 37°C room
- 15 mL falcon tubes (~x15)
- eppendorf tubes (1 mL)
- 50% glycerol
- 0.1 M CaCl2
- waste bucket with Vircon (kill bacteria)
- ice buckets
Protocol:
- inoculate 1% inoculum from overnight culture
- 1 mL for 100 mL medium
- 2 separate 250 mL flasks each with 50 mL LB
- 0.5 mL of culture- grow with shaking until ~ 0.375 OD (at 600 nm) - we went a bit over, but other protocols say this is ok
- put flasks on ice for 10 minutes
- put 10 mL of inoculum into 15 mL falcon tubes (repeat to use up all the inoculum)
- centrifuge @ 5K for 10 minutes @ 4°C
- discard supernatant and resuspend pellet in 2 mL of 0.1 M CaCl2
- chill on ice for 20 mins
- centrifuge @ 5K for 10 minutes @ 4°C
- discard supernatant and resuspend each pellet in 0.28 mL of 0.1 M CaCl2 and 0.12 mL of 50% glycerol - you can also prepare a solution of CaCl2 and glycerol before hand
- aliquot 100 μL of resuspension into sterile eppendorfs
- store at -80°C