Berkeley LBL/CompetentCell

Revision as of 21:18, 17 October 2007 by Joyxi (Talk | contribs)

(diff) ← Older revision | Latest revision (diff) | Newer revision → (diff)

KCM Chemical Competent Cell Production Protocol

Solutions:

LB (250mL)

TSS (100mL):

       10g PEG 3350

       5mL DMSO

       2mL 1M MgCl2

       Bring to 100mL with LB

       Filter, sterilize & chill at 4ºC

KCM:

       0.5M KCl

       0.15M CaCl2

       0.25M MgCl2

Protocol (for 250 aliquots):

1. Grow overnight culture from single colony

2. Inoculate 1:100 in 250mL LB, grow to OD 0.4-0.5

3. Chill on ice for 20min

4. Centrifuge at 8000g for 8min, decant supernatant

5. Resuspend in 25mL ice cold TSS using a 25mL pipet.

6. Make sure there are no clumps.

7. Aliquot 100ul into each tube. Freeze in liquid nitrogen and store at -80ºC