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This protocol should be doubled up - it is almost the same amount of work and you can get some 80 tubes.
- Inoculate colony O/N in 3mL LB+antibiotics at 30˚C shaker
- Transfer O/N cultures to 200mL LB in sterile 500mL flask and shake at 250rpm until the OD is 0.3 (4-5h)
- Spin in sterile screw cap tubes at 4˚C, 5000 rpm for 10min. Check to make sure cells are pelleted - if not repeat at higher speed
- Aspirate supernatant, resuspend pellet in 20mL ice cold 1mM HEPES pH7 (sterile filtered), re-spin. Repeat this step 2 more times.
- After aspirating, resuspend pellet in 2mL ice cold 10% glycerol (sterile filtered)
- ASAP dispense in 40ul aliquots in pre-chilled, sterile eppis, freeze in liquid nitrogen and store at -70˚C