Transforming chemically competent cells (Inoue) - Revision history

Vaishnavi Ananth: /* BioStream version */

Vaishnavi Ananth — Fri, 20 Nov 2009 05:32:46 GMT

BioStream version

←Older revision		Revision as of 05:32, 20 November 2009
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	Achieved efficiency was 3 x 10<sup>7</sup> per microgram. A control transformation with Invitrogen cells was at 1.2 x 10<sup>8</sup> per microgram.		Achieved efficiency was 3 x 10<sup>7</sup> per microgram. A control transformation with Invitrogen cells was at 1.2 x 10<sup>8</sup> per microgram.

-	==~~BioStream~~ version==	+	==BioCoder version==
-	Following is the Transforming chemically competent cells (Inoue) protocol in ~~BioStream~~, a high-level programming language for expressing biology protocols. What you see here is the auto-generated text ouput of the protocol that was coded up in ~~BioStream~~ (see Source code). More information about ~~BioStream~~ can be found on my home page. Feel free to mail me your comments/ suggestions.[[User:Vaishnavi Ananth\|Vaishnavi]]	+	Following is the Transforming chemically competent cells (Inoue) protocol in BioCoder, a high-level programming language for expressing biology protocols. What you see here is the auto-generated text ouput of the protocol that was coded up in BioCoder (see Source code). More information about BioCoder can be found on my home page. Feel free to mail me your comments/ suggestions.[[User:Vaishnavi Ananth\|Vaishnavi]]
	====Text Output====		====Text Output====
	[[Transforming chemically competent cells (Inoue) protocol]]		[[Transforming chemically competent cells (Inoue) protocol]]

Vaishnavi Ananth at 09:00, 23 October 2009

Vaishnavi Ananth — Fri, 23 Oct 2009 09:00:08 GMT

←Older revision		Revision as of 09:00, 23 October 2009
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	Achieved efficiency was 3 x 10<sup>7</sup> per microgram. A control transformation with Invitrogen cells was at 1.2 x 10<sup>8</sup> per microgram.		Achieved efficiency was 3 x 10<sup>7</sup> per microgram. A control transformation with Invitrogen cells was at 1.2 x 10<sup>8</sup> per microgram.
		+
		+	==BioStream version==
		+	Following is the Transforming chemically competent cells (Inoue) protocol in BioStream, a high-level programming language for expressing biology protocols. What you see here is the auto-generated text ouput of the protocol that was coded up in BioStream (see Source code). More information about BioStream can be found on my home page. Feel free to mail me your comments/ suggestions.[[User:Vaishnavi Ananth\|Vaishnavi]]
		+	====Text Output====
		+	[[Transforming chemically competent cells (Inoue) protocol]]
		+	====Source Code====
		+	[[Transforming chemically competent cells (Inoue) protocol - source code]]

	[[Category:Protocol]]		[[Category:Protocol]]
	[[Category:Escherichia coli]]		[[Category:Escherichia coli]]

Reshma P. Shetty at 22:46, 5 December 2006

Reshma P. Shetty — Tue, 05 Dec 2006 22:46:16 GMT

←Older revision		Revision as of 22:46, 5 December 2006
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	Achieved efficiency was 3 x 10<sup>7</sup> per microgram. A control transformation with Invitrogen cells was at 1.2 x 10<sup>8</sup> per microgram.		Achieved efficiency was 3 x 10<sup>7</sup> per microgram. A control transformation with Invitrogen cells was at 1.2 x 10<sup>8</sup> per microgram.

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

Boyuanzhu: /* Experimental results */

Boyuanzhu — Thu, 22 Jun 2006 15:11:45 GMT

Experimental results

←Older revision		Revision as of 15:11, 22 June 2006
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	* Incubating for 1/2 hour on ice had a positive effect, perhaps 1.5 to 2x efficiency gain.		* Incubating for 1/2 hour on ice had a positive effect, perhaps 1.5 to 2x efficiency gain.
	* Heat shock of 0 or 15 s rather than 30 s reduced efficiency about 8x		* Heat shock of 0 or 15 s rather than 30 s reduced efficiency about 8x
-	* Heat shock at 30 s or 60 s gave approximately similar results.	+	* Heat shock at 30 s or 60 s gave approximately similar results. (*Edit: 50s is preferable)

	Achieved efficiency was 3 x 10<sup>7</sup> per microgram. A control transformation with Invitrogen cells was at 1.2 x 10<sup>8</sup> per microgram.		Achieved efficiency was 3 x 10<sup>7</sup> per microgram. A control transformation with Invitrogen cells was at 1.2 x 10<sup>8</sup> per microgram.

Tk at 18:41, 18 April 2006

Tk — Tue, 18 Apr 2006 18:41:26 GMT

←Older revision		Revision as of 18:41, 18 April 2006
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	#Spread 100-300 μl onto a plate made with appropriate antibiotic.		#Spread 100-300 μl onto a plate made with appropriate antibiotic.
	#Grow overnight at 37°C.		#Grow overnight at 37°C.
		+
		+	==Experimental results==
		+	First attempt varied several parameters: incubation time on ice prior to heat shock, heat shock length, addition of DTT at 20mM.
		+
		+	* DTT appeared to have little effect when added during transformation.
		+	* Incubating for 1/2 hour on ice had a positive effect, perhaps 1.5 to 2x efficiency gain.
		+	* Heat shock of 0 or 15 s rather than 30 s reduced efficiency about 8x
		+	* Heat shock at 30 s or 60 s gave approximately similar results.
		+
		+	Achieved efficiency was 3 x 10<sup>7</sup> per microgram. A control transformation with Invitrogen cells was at 1.2 x 10<sup>8</sup> per microgram.
		+

	[[Category:E.Coli Protocol]]		[[Category:E.Coli Protocol]]

Reshma P. Shetty: /* Method */

Reshma P. Shetty — Wed, 12 Apr 2006 12:28:40 GMT

Method

←Older revision		Revision as of 12:28, 12 April 2006
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	#Incubate on ice for 30 minutes		#Incubate on ice for 30 minutes
	#*Note: If you are in a rush, you can shorten this incubation time to 5-10 min.		#*Note: If you are in a rush, you can shorten this incubation time to 5-10 min.
-	#Incubate cells for 30 seconds at ~~<math>~~42~~^o</math>~~C.	+	#Incubate cells for 30 seconds at 42°C.
	#Incubate cells on ice for 2 min.		#Incubate cells on ice for 2 min.
	#Add 4 volumes of room temperature [[SOC]] (not critical)		#Add 4 volumes of room temperature [[SOC]] (not critical)
-	#Incubate for 1 hour at ~~<math>~~37~~^o</math>~~C on shaker.	+	#Incubate for 1 hour at 37°C on shaker.
	#*Note: Can also save some time here by reducing incubation to ~45 min.		#*Note: Can also save some time here by reducing incubation to ~45 min.
	#*Note: Step can be eliminated if plating on Amp plates, but not most other antibiotics		#*Note: Step can be eliminated if plating on Amp plates, but not most other antibiotics
	#Spread 100-300 μl onto a plate made with appropriate antibiotic.		#Spread 100-300 μl onto a plate made with appropriate antibiotic.
-	#Grow overnight at 37 °C.	+	#Grow overnight at 37°C.

	[[Category:E.Coli Protocol]]		[[Category:E.Coli Protocol]]

Tk at 01:29, 12 April 2006

Tk — Wed, 12 Apr 2006 01:29:35 GMT

New page

''Also see [[Preparing chemically competent cells (Inoue)]]''

==Method==
#Thaw 25 - 200 μl TB buffer cells on ice. Do not use glass tubes, which adsorb DNA.
#Add DNA, pipette gently to mix (keep volume of DNA less than 5% of the cell volume)
#Incubate on ice for 30 minutes
#*Note: If you are in a rush, you can shorten this incubation time to 5-10 min.
#Incubate cells for 30 seconds at <math>42^o</math>C.
#Incubate cells on ice for 2 min.
#Add 4 volumes of room temperature [[SOC]] (not critical)
#Incubate for 1 hour at <math>37^o</math>C on shaker.
#*Note: Can also save some time here by reducing incubation to ~45 min.
#*Note: Step can be eliminated if plating on Amp plates, but not most other antibiotics
#Spread 100-300 μl onto a plate made with appropriate antibiotic.
#Grow overnight at 37 °C.

[[Category:E.Coli Protocol]]