IGEM:UC Berkeley/2006/KCM cells

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  1. Overnight culture in 10 mL LB, fresh colony.
  2. Dilute 1:100 in 500 mL LB, grow to OD .4-.5, takes between 2-3 hours. Shake on ice to stop the growth before spin.
  3. Spin in prechilled bottle at 3500rpm for 15min.
  4. Do the rest in the cold room or on ice bucket: resuspend in 25-50mL ICE-COLD TSS with prechilled pippette.

TSS(100mL of LB):
In LB

 10% PEG-3350 (10g)
 5% DMSO (5mL)
 20mM MgCl2 (2mL from a 1M stock)
 pH 6.5
 filter sterilize
  1. Aliquot into 200uL tube using multipipette.
  2. Freeze cells in liquid nitrogen bath.

Prechill

 rotor
 25-50mL pipette for cell suspension
 500mL centrifuge bottle
 whole beaker of eppendorf tubes
 5mL multipipette
 TSS solution
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