Preparing chemically competent cells (Inoue)

Materials

 * Plate of cells streaked for single colonies
 * SOB
 * Ice
 * TB buffer
 * DMSO
 * Dry Ice (or liquid nitrogen)

Glassware & equipment

 * 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

Preparation

 * 1) 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.
 * 2) Grow to  an OD of 0.6 with vigorous shaking (200-250 rpm) at 18 degrees (important).  This is slow -- approximately 35-40 hours.
 * 3) Prechill the centrifuge to 4 degrees
 * 4) Remove from the incubator and place on ice for 10 minutes
 * 5) Transfer to two 220 ml centrifuge tubes and spin at 3220 x g for 10 minutes at 4 degrees
 * 6) 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.
 * 7) Place on ice for 10 minutes
 * 8) Spin down as above.
 * 9) While spinning, add 1.4 ml of DMSO to 18.6 ml of TB  (7% DMSO mixture)
 * 10) Resuspend each pellet in 20 ml of cold TB-DMSO mixture
 * 11) Incubate on ice for 10 minutes
 * 12) Dispense cells into pre-chilled tubes
 * 13) Freeze cells in a dry ice / ethanol bath and store at -80 degrees indefinitely

Thoughts on improvements

 * "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.
 * My lab uses LB for instead of SOB media...it seems to work fine for them--mel 18:10, 14 June 2007 (EDT)

Related topics & references

 * Preparing chemically competent cells
 * Preparing TSS buffer
 * Transforming chemically competent cells''
 * Preparing electrocompetent cells
 * Electroporation
 * TB buffer
 * Transforming chemically competent cells (Inoue)
 * Bacterial cell culture

Original protocol from Inoue et al. Inoue90. Useful comments and speculation about reducing agents in Hengen96.


 * 1) Inoue90 pmid=2265755
 * 2) Hengen96 pmid=8851666