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
