Preparing chemically competent cells (Inoue): Difference between revisions
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{{back to protocols}} | |||
==Materials== | ==Materials== | ||
* Plate of cells streaked for single colonies | * Plate of cells streaked for single colonies | ||
* [[SOB | * [[SOB]] | ||
* Ice | * Ice | ||
* [[TB buffer]] | * [[TB buffer]] | ||
Line 8: | Line 9: | ||
==Glassware & equipment== | ==Glassware & equipment== | ||
* 2 liter erlenmeyer flask | * 2 liter erlenmeyer flask (no detergent residue, rinse with 70% ethanol and DI water) | ||
* 220 ml conical centrifuge tubes BD 35 2075 | * 220 ml conical centrifuge tubes BD 35 2075 | ||
* Eppendorf 5410R refrigerated centrifuge with conical adapters | * Eppendorf 5410R refrigerated centrifuge with conical adapters | ||
==Preparation== | ==Preparation== | ||
# Pick a 10 -12 large single colonies (2-3 mm dia) from your source plate and inoculate 500 ml of SOB medium in a 2 liter flask. | # 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 | # 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 | # Prechill the centrifuge to 4 degrees | ||
# Remove from the incubator and place on ice for 10 minutes | # Remove from the incubator and place on ice for 10 minutes | ||
Line 21: | Line 22: | ||
# Place on ice for 10 minutes | # Place on ice for 10 minutes | ||
# Spin down as above. | # Spin down as above. | ||
# Resuspend each pellet in 20 ml of cold TB | # 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 | # Incubate on ice for 10 minutes | ||
# Dispense cells into pre-chilled tubes | # Dispense cells into pre-chilled tubes | ||
# Freeze cells in a dry ice / ethanol bath and store at -80 degrees indefinitely | # 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--[[User:Melissali|mel]] 18:10, 14 June 2007 (EDT) | |||
==Related topics & references== | ==Related topics & references== | ||
Line 33: | Line 43: | ||
*[[Electrocompetent cells|Preparing electrocompetent cells]] | *[[Electrocompetent cells|Preparing electrocompetent cells]] | ||
*[[Electroporation]] | *[[Electroporation]] | ||
*[[TB buffer]] | |||
*[[Transforming chemically competent cells (Inoue)]] | |||
*[[Bacterial cell culture]] | *[[Bacterial cell culture]] | ||
Original protocol from Inoue et al. <cite> Inoue90 </cite>. | Original protocol from Inoue et al. <cite> Inoue90 </cite>. Useful comments and speculation about reducing agents in <cite>Hengen96</cite>. | ||
<biblio> | <biblio> | ||
#Inoue90 pmid=2265755 | #Inoue90 pmid=2265755 | ||
#Hengen96 pmid=8851666 | |||
</biblio> | </biblio> | ||
[[Category: | [[Category:Protocol]] | ||
[[Category:Escherichia coli]] |
Latest revision as of 10:09, 23 February 2009
back to protocols | ||
Materials
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
- 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
- Electroporation
- TB buffer
- Transforming chemically competent cells (Inoue)
- Bacterial cell culture
Original protocol from Inoue et al. [1]. Useful comments and speculation about reducing agents in [2].
- 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.