IGEM:UC Berkeley/2006/KCM cells: Difference between revisions
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#Spin in prechilled bottle at 3500rpm for 15min. | #Spin in prechilled bottle at 3500rpm for 15min. | ||
#Do the rest in the cold room or on ice bucket: resuspend in 25-50mL ICE-COLD TSS with prechilled pippette.<br> | #Do the rest in the cold room or on ice bucket: resuspend in 25-50mL ICE-COLD TSS with prechilled pippette.<br> | ||
'''TSS(100mL of | '''TSS(100mL of [[LB]]):''' | ||
10% PEG-3350 (10g) | 10% PEG-3350 (10g) | ||
5% DMSO (5mL) | 5% DMSO (5mL) | ||
Revision as of 09:39, 29 June 2006
- Overnight culture in 10 mL LB, fresh colony.
- 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.
- Spin in prechilled bottle at 3500rpm for 15min.
- 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):
10% PEG-3350 (10g) 5% DMSO (5mL) 20mM MgCl2 (2mL from a 1M stock) pH 6.5
- Aliquot into 200uL tube using multipipette.
- 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
