Preparing chemically competent cells - Revision history

Yanglong Zhu: /* Notes */

Yanglong Zhu — Thu, 04 Apr 2013 18:11:46 GMT

Notes

←Older revision		Revision as of 18:11, 4 April 2013
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	==Notes==		==Notes==
-	*Note1: CT Chung paper recommends long storage at -70˚C, while people have been using a wide range of temperatures from -20˚C to -140˚C for long term storage.	+	*Note1: CT Chung paper recommends long storage of TSS competent cells at -70˚C, while people have been using a wide range of temperatures from -20˚C to -140˚C for long term storage.

	==Related topics & References==		==Related topics & References==

Yanglong Zhu: /* Preparation */

Yanglong Zhu — Thu, 04 Apr 2013 18:10:42 GMT

Preparation

←Older revision		Revision as of 18:10, 4 April 2013
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	#It is a good idea to run a positive control on the cells.		#It is a good idea to run a positive control on the cells.
	#*The [[Endy Lab]] is trying to use a standard positive control to better compare (and hopefully improve) the transformation efficiencies in the lab, you can check it out [[Endy:Standard transformation positive control\|here]].		#*The [[Endy Lab]] is trying to use a standard positive control to better compare (and hopefully improve) the transformation efficiencies in the lab, you can check it out [[Endy:Standard transformation positive control\|here]].
		+
		+	==Notes==
		+	*Note1: CT Chung paper recommends long storage at -70˚C, while people have been using a wide range of temperatures from -20˚C to -140˚C for long term storage.

	==Related topics & References==		==Related topics & References==

Roberto Rosati at 02:39, 13 April 2010

Roberto Rosati — Tue, 13 Apr 2010 02:39:57 GMT

←Older revision		Revision as of 02:39, 13 April 2010
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	#Add 100 μl aliquots to your chilled eppendorfs and store at <math>-80^o</math>C.		#Add 100 μl aliquots to your chilled eppendorfs and store at <math>-80^o</math>C.
	#*The original paper <cite> chung </cite> suggests freezing the cells immediately using a dry ice bath. I ([[Barry Canton\|BC]]) have used liquid nitrogen quite successfully instead of dry ice. Simply placing the cells at <math>-80^o</math>C also seems to work well ([[User:Jkm\|Jkm]])		#*The original paper <cite> chung </cite> suggests freezing the cells immediately using a dry ice bath. I ([[Barry Canton\|BC]]) have used liquid nitrogen quite successfully instead of dry ice. Simply placing the cells at <math>-80^o</math>C also seems to work well ([[User:Jkm\|Jkm]])
		+	#*If you run a control every time you clone (i.e. a vector-only ligation), you can as well freeze cells in 200 μl aliquots. Unused cells can be frozen back once and reused, albeit with some loss of competence.
	#It is a good idea to run a positive control on the cells.		#It is a good idea to run a positive control on the cells.
	#*The [[Endy Lab]] is trying to use a standard positive control to better compare (and hopefully improve) the transformation efficiencies in the lab, you can check it out [[Endy:Standard transformation positive control\|here]].		#*The [[Endy Lab]] is trying to use a standard positive control to better compare (and hopefully improve) the transformation efficiencies in the lab, you can check it out [[Endy:Standard transformation positive control\|here]].

Torsten Waldminghaus at 17:08, 23 February 2009

Torsten Waldminghaus — Mon, 23 Feb 2009 17:08:30 GMT

←Older revision		Revision as of 17:08, 23 February 2009
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		+	{{back to protocols}}
	==Materials==		==Materials==
	*Plate of cells to be made competent		*Plate of cells to be made competent

Reshma P. Shetty at 21:21, 8 May 2007

Reshma P. Shetty — Tue, 08 May 2007 21:21:39 GMT

←Older revision		Revision as of 21:21, 8 May 2007
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	[[Category:Protocol]]		[[Category:Protocol]]
		+	[[Category:In vivo]]
	[[Category:Escherichia coli]]		[[Category:Escherichia coli]]

Jason R. Kelly: /* Preparation */

Jason R. Kelly — Mon, 26 Feb 2007 20:15:33 GMT

Preparation

←Older revision		Revision as of 20:15, 26 February 2007
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	#Add 100 μl aliquots to your chilled eppendorfs and store at <math>-80^o</math>C.		#Add 100 μl aliquots to your chilled eppendorfs and store at <math>-80^o</math>C.
	#*The original paper <cite> chung </cite> suggests freezing the cells immediately using a dry ice bath. I ([[Barry Canton\|BC]]) have used liquid nitrogen quite successfully instead of dry ice. Simply placing the cells at <math>-80^o</math>C also seems to work well ([[User:Jkm\|Jkm]])		#*The original paper <cite> chung </cite> suggests freezing the cells immediately using a dry ice bath. I ([[Barry Canton\|BC]]) have used liquid nitrogen quite successfully instead of dry ice. Simply placing the cells at <math>-80^o</math>C also seems to work well ([[User:Jkm\|Jkm]])
-
	#It is a good idea to run a positive control on the cells.		#It is a good idea to run a positive control on the cells.
-	The [[Endy Lab]] is trying to use a standard positive control to better compare (and hopefully improve) the transformation efficiencies in the lab, you can check it out [[Endy:Standard transformation positive control\|here]].	+	#*The [[Endy Lab]] is trying to use a standard positive control to better compare (and hopefully improve) the transformation efficiencies in the lab, you can check it out [[Endy:Standard transformation positive control\|here]].

	==Related topics & References==		==Related topics & References==

Jason R. Kelly: /* Preparation */

Jason R. Kelly — Mon, 26 Feb 2007 20:15:16 GMT

Preparation

←Older revision		Revision as of 20:15, 26 February 2007
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	#*The original paper <cite> chung </cite> suggests freezing the cells immediately using a dry ice bath. I ([[Barry Canton\|BC]]) have used liquid nitrogen quite successfully instead of dry ice. Simply placing the cells at <math>-80^o</math>C also seems to work well ([[User:Jkm\|Jkm]])		#*The original paper <cite> chung </cite> suggests freezing the cells immediately using a dry ice bath. I ([[Barry Canton\|BC]]) have used liquid nitrogen quite successfully instead of dry ice. Simply placing the cells at <math>-80^o</math>C also seems to work well ([[User:Jkm\|Jkm]])

-	*The [[Endy Lab]] is trying to use a standard positive control to better compare (and hopefully improve) the transformation efficiencies in the lab, you can check it out [[Endy:Standard transformation positive control\|here]].	+	#It is a good idea to run a positive control on the cells.
		+	The [[Endy Lab]] is trying to use a standard positive control to better compare (and hopefully improve) the transformation efficiencies in the lab, you can check it out [[Endy:Standard transformation positive control\|here]].

	==Related topics & References==		==Related topics & References==

Jason R. Kelly: /* Preparation */

Jason R. Kelly — Mon, 26 Feb 2007 20:14:38 GMT

Preparation

←Older revision		Revision as of 20:14, 26 February 2007
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	#Add 100 μl aliquots to your chilled eppendorfs and store at <math>-80^o</math>C.		#Add 100 μl aliquots to your chilled eppendorfs and store at <math>-80^o</math>C.
	#*The original paper <cite> chung </cite> suggests freezing the cells immediately using a dry ice bath. I ([[Barry Canton\|BC]]) have used liquid nitrogen quite successfully instead of dry ice. Simply placing the cells at <math>-80^o</math>C also seems to work well ([[User:Jkm\|Jkm]])		#*The original paper <cite> chung </cite> suggests freezing the cells immediately using a dry ice bath. I ([[Barry Canton\|BC]]) have used liquid nitrogen quite successfully instead of dry ice. Simply placing the cells at <math>-80^o</math>C also seems to work well ([[User:Jkm\|Jkm]])
		+
		+	*The [[Endy Lab]] is trying to use a standard positive control to better compare (and hopefully improve) the transformation efficiencies in the lab, you can check it out [[Endy:Standard transformation positive control\|here]].

	==Related topics & References==		==Related topics & References==

Epsilon11: /* Preparation */

Epsilon11 — Sun, 18 Feb 2007 13:08:46 GMT

Preparation

←Older revision		Revision as of 13:08, 18 February 2007
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	#Grow a 5ml [[Bacterial cell culture\|overnight culture]] of cells in LB media. In the morning, dilute this culture back into 25-50ml of fresh LB media in a 200ml conical flask. You should aim to dilute the overnight culture by at least 1/100.		#Grow a 5ml [[Bacterial cell culture\|overnight culture]] of cells in LB media. In the morning, dilute this culture back into 25-50ml of fresh LB media in a 200ml conical flask. You should aim to dilute the overnight culture by at least 1/100.
	#Grow the diluted culture to an OD<sub>600</sub> of 0.2 - 0.5. (You will get a very small pellet if you grow 25ml to OD<sub>600</sub> 0.2)		#Grow the diluted culture to an OD<sub>600</sub> of 0.2 - 0.5. (You will get a very small pellet if you grow 25ml to OD<sub>600</sub> 0.2)
-	#Put eppendorf tubes on ice now so that they are cold when cells are aliquoted into them later. If your culture is ~~Xml~~, you will need X tubes. At this point you should also make sure that your [[TSS]] is being chilled (it should be stored at 4C but if you have just made it fresh then put it in an ice bath).	+	#Put eppendorf tubes on ice now so that they are cold when cells are aliquoted into them later. If your culture is X ml, you will need X tubes. At this point you should also make sure that your [[TSS]] is being chilled (it should be stored at <math>4^o</math>C but if you have just made it fresh then put it in an ice bath).
	#Split the culture into two 50ml falcon tubes and incubate on ice for 10 min.		#Split the culture into two 50ml falcon tubes and incubate on ice for 10 min.
	'''All subsequent steps should be carried out at <math>4^o</math>C and the cells should be kept on ice wherever possible'''		'''All subsequent steps should be carried out at <math>4^o</math>C and the cells should be kept on ice wherever possible'''

Reshma P. Shetty at 22:43, 5 December 2006

Reshma P. Shetty — Tue, 05 Dec 2006 22:43:11 GMT

←Older revision		Revision as of 22:43, 5 December 2006
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	</biblio>		</biblio>

-	[[Category:~~E.Coli~~ Protocol]]	+	[[Category:Protocol]]
		+	[[Category:Escherichia coli]]