Preparing chemically competent cells (Inoue)
Jump to navigationJump to search
|back to protocols|
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
- 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
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
- TB buffer
- Transforming chemically competent cells (Inoue)
- Bacterial cell culture
- Inoue H, Nojima H, and Okayama H. High efficiency transformation of Escherichia coli with plasmids. Gene. 1990 Nov 30;96(1):23-8. DOI:10.1016/0378-1119(90)90336-p |
- Hengen PN. Methods and reagents. preparing ultra-competent Escherichia coli. Trends Biochem Sci. 1996 Feb;21(2):75-6.