Preparing chemically competent cells (Inoue) - Revision history

Torsten Waldminghaus at 17:09, 23 February 2009

Torsten Waldminghaus — Mon, 23 Feb 2009 17:09:01 GMT

←Older revision		Revision as of 17:09, 23 February 2009
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		+	{{back to protocols}}
	==Materials==		==Materials==
	* Plate of cells streaked for single colonies		* Plate of cells streaked for single colonies

Melissali — Thu, 14 Jun 2007 22:10:16 GMT

Thoughts on improvements

←Older revision		Revision as of 22:10, 14 June 2007
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	* The Hanahan protocol specifies dry pure DMSO, while Inoue says it doesn't make a difference. Let's see.		* 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.		* 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==

Reshma P. Shetty — Tue, 05 Dec 2006 22:45:05 GMT

Tk — Tue, 25 Apr 2006 19:03:36 GMT

Thoughts on improvements

←Older revision		Revision as of 19:03, 25 April 2006
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	* Length of time on ice prior to transformation may make a big difference		* 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.		* 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.

	==Related topics & references==		==Related topics & references==

Tk — Tue, 18 Apr 2006 18:46:13 GMT

Thoughts on improvements

←Older revision		Revision as of 18:46, 18 April 2006
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	==Thoughts on improvements==		==Thoughts on improvements==
	* "Methods in Yeast Genetics" book (Amberg05) suggests growth the SOB + 300 mM NaCl		* "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		* 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		* 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.

	==Related topics & references==		==Related topics & references==

Tk — Tue, 18 Apr 2006 18:44:37 GMT

←Older revision		Revision as of 18:44, 18 April 2006
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	# 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
		+	* 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
		+

	==Related topics & references==		==Related topics & references==

Tk — Fri, 14 Apr 2006 23:09:19 GMT

←Older revision		Revision as of 23:09, 14 April 2006
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	*[[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>

Tk — Fri, 14 Apr 2006 20:37:44 GMT

←Older revision		Revision as of 20:37, 14 April 2006
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	# 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)
-	~~# Add~~ DMSO ~~to a final concentration of 7%~~	+	# 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

Tk — Fri, 14 Apr 2006 18:07:46 GMT

Preparation

←Older revision		Revision as of 18:07, 14 April 2006
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	==Preparation==		==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.	+	# 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)	+	# 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

Tk — Wed, 12 Apr 2006 14:43:45 GMT