Revision as of 10:07, 8 July 2011

Competent cells preparation (by Inoue)

2 liter erlenmeyer flask (no detergent residue, rinse with 70% ethanol and DI water)
220 ml conical centrifuge tubes BD 35 2075
Eppendorf 5410R refrigerated centrifuge with conical adapters

Pick a 10 - 12 large single colonies (2-3 mm dia) from your source plate and inoculate 500 ml of sterile SOB medium (do not use LB) in a 2 liter flask. Save some medium as an OD blank.
Grow to an OD of 0.6 with vigorous shaking (200-250 rpm) at 18 degrees (important). This is slow -- approximately 35-40 hours.
Prechill the centrifuge to 4 degrees
Remove from the incubator and place on ice for 10 minutes
Transfer to two 220 ml centrifuge tubes and spin at 3220 x g for 10 minutes at 4 degrees
Drain the medium and resuspend each pellet first in 5 ml of ice cold TB. Add an additional 75 ml of cold TB buffer and resuspend.
Place on ice for 10 minutes
Spin down as above.
While spinning, add 1.4 ml of DMSO to 18.6 ml of TB (7% DMSO mixture)
Resuspend each pellet in 20 ml of cold TB-DMSO mixture
Incubate on ice for 10 minutes
Dispense cells into pre-chilled tubes
Freeze cells in a dry ice / ethanol bath and store at -80 degrees indefinitely

"Methods in Yeast Genetics" book (Amberg05) suggests growth the SOB + 300 mM NaCl
They also control pH at 7.5, which may be a major issue
Centrifuging in flat bottom centrifuge tubes may make pellet resuspension easier and less damaging
Length of time on ice prior to transformation may make a big difference
The Hanahan protocol specifies dry pure DMSO, while Inoue says it doesn't make a difference. Let's see.
Warm plates for growing cells after transformation are claimed to be 2x to 4x more efficient.

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