Endy:Colony PCR - Revision history

Vaishnavi Ananth: /* BioStream version */

Vaishnavi Ananth — Thu, 19 Nov 2009 09:44:44 GMT

BioStream version

←Older revision		Revision as of 09:44, 19 November 2009
Line 25:		Line 25:
	*For shorter amplicons, under ~1kb, this can be shortened judiciously.		*For shorter amplicons, under ~1kb, this can be shortened judiciously.

-	==~~BioStream~~ version==	+	==BioCoder version==
-	Following is the Endy:Colony PCR 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 Endy:Colony PCR 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====
	[[Endy:Colony PCR protocol]]		[[Endy:Colony PCR protocol]]

Vaishnavi Ananth at 07:12, 23 September 2009

Vaishnavi Ananth — Wed, 23 Sep 2009 07:12:43 GMT

←Older revision		Revision as of 07:12, 23 September 2009
Line 24:		Line 24:
	*For long amplicons, X = 1 minute + 2.5 s per 100bp		*For long amplicons, X = 1 minute + 2.5 s per 100bp
	*For shorter amplicons, under ~1kb, this can be shortened judiciously.		*For shorter amplicons, under ~1kb, this can be shortened judiciously.
		+
		+	==BioStream version==
		+	Following is the Endy:Colony PCR 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====
		+	[[Endy:Colony PCR protocol]]
		+	====Source Code====
		+	[[Endy:Colony PCR protocol - source code]]

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

Michael P. Killoran at 19:22, 22 April 2009

Michael P. Killoran — Wed, 22 Apr 2009 19:22:56 GMT

←Older revision		Revision as of 19:22, 22 April 2009
Line 5:		Line 5:

	===Reaction Mix===		===Reaction Mix===
-	Use the following reaction mix for each PCR ~~reaction~~:	+	Use the following reaction mix for each PCR:
	*1 μl 10x Thermo polymerase buffer		*1 μl 10x Thermo polymerase buffer
	*1 μl 10x dNTPs (10x = 2.5 mM each dNTP)		*1 μl 10x dNTPs (10x = 2.5 mM each dNTP)

Nishant M. Bhat: /* Protocol */

Nishant M. Bhat — Wed, 13 Jun 2007 13:56:47 GMT

Protocol

←Older revision		Revision as of 13:56, 13 June 2007
Line 2:		Line 2:
	==Protocol==		==Protocol==
	*Use a sterile toothpick or pipet tip to resuspend a plated colony in 50 μl sterile water.		*Use a sterile toothpick or pipet tip to resuspend a plated colony in 50 μl sterile water.
		+	*store the colony resuspension at 4C so you can start cultures if necessary (should be OK for a couple days, if you need it to last longer you should use an [[Index plate]].

	===Reaction Mix===		===Reaction Mix===

Reshma P. Shetty at 17:42, 20 April 2007

Reshma P. Shetty — Fri, 20 Apr 2007 17:42:43 GMT

←Older revision		Revision as of 17:42, 20 April 2007
Line 23:		Line 23:
	*For long amplicons, X = 1 minute + 2.5 s per 100bp		*For long amplicons, X = 1 minute + 2.5 s per 100bp
	*For shorter amplicons, under ~1kb, this can be shortened judiciously.		*For shorter amplicons, under ~1kb, this can be shortened judiciously.
		+
		+	[[Category:Escherichia coli]] [[Category:Protocol]]

Barry Canton: /* PCR protocol */

Barry Canton — Tue, 09 Aug 2005 14:41:24 GMT

PCR protocol

←Older revision		Revision as of 14:41, 9 August 2005
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	*Cycle 35 times:		*Cycle 35 times:
	**95 C for 30 s (melting)		**95 C for 30 s (melting)
-	**53 C for 30 s (annealing)	+	**53 C (or whatever temperature is appropriate) for 30 s (annealing)
	**72 C for X s (elongation)		**72 C for X s (elongation)
	*72 C for 10 minutes (final elongation)		*72 C for 10 minutes (final elongation)

Barry Canton at 16:22, 14 July 2005

Barry Canton — Thu, 14 Jul 2005 16:22:13 GMT

←Older revision		Revision as of 16:22, 14 July 2005
Line 20:		Line 20:
	**72 C for X s (elongation)		**72 C for X s (elongation)
	*72 C for 10 minutes (final elongation)		*72 C for 10 minutes (final elongation)
-	*4 C forever ~~(hold)~~	+	*4 C forever
	*For long amplicons, X = 1 minute + 2.5 s per 100bp		*For long amplicons, X = 1 minute + 2.5 s per 100bp
	*For shorter amplicons, under ~1kb, this can be shortened judiciously.		*For shorter amplicons, under ~1kb, this can be shortened judiciously.

Barry Canton: /* Reaction Mix */

Barry Canton — Thu, 14 Jul 2005 16:14:15 GMT

Reaction Mix

←Older revision		Revision as of 16:14, 14 July 2005
Line 9:		Line 9:
	*0.15 μl 40 μM FWD primer		*0.15 μl 40 μM FWD primer
	*0.15 μl 40 μM REV primer		*0.15 μl 40 μM REV primer
-	*0.1 μl Polymerase	+	*0.1 μl Polymerase (taq or vent)
	*6.6 μl H<sub>2</sub>O		*6.6 μl H<sub>2</sub>O
	*1.0 μl template suspension		*1.0 μl template suspension

Barry Canton at 16:11, 14 July 2005

Barry Canton — Thu, 14 Jul 2005 16:11:21 GMT

New page

''See [[Colony PCR]] for general information about this protocol and other variants''
==Protocol==
*Use a sterile toothpick or pipet tip to resuspend a plated colony in 50 μl sterile water.

===Reaction Mix===
Use the following reaction mix for each PCR reaction:
*1 μl 10x Thermo polymerase buffer
*1 μl 10x dNTPs (10x = 2.5 mM each dNTP)
*0.15 μl 40 μM FWD primer
*0.15 μl 40 μM REV primer
*0.1 μl Polymerase
*6.6 μl H<sub>2</sub>O
*1.0 μl template suspension

===PCR protocol===
*95 C for 6 minutes (disrupt cells, separate DNA)
*Cycle 35 times:
**95 C for 30 s (melting)
**53 C for 30 s (annealing)
**72 C for X s (elongation)
*72 C for 10 minutes (final elongation)
*4 C forever (hold)
*For long amplicons, X = 1 minute + 2.5 s per 100bp
*For shorter amplicons, under ~1kb, this can be shortened judiciously.