PCR Overlap Extension - Revision history

Christopher C Vanlang at 10:30, 22 December 2011

Christopher C Vanlang — Thu, 22 Dec 2011 10:30:19 GMT

←Older revision		Revision as of 10:30, 22 December 2011
Line 38:		Line 38:
	[[Category:DNA]]		[[Category:DNA]]
	[[Category:In vitro]]		[[Category:In vitro]]
		+	[[Category:PCR]]

Michael A. Speer: /* Procedure */

Michael A. Speer — Thu, 06 Oct 2011 00:55:13 GMT

Procedure

←Older revision		Revision as of 00:55, 6 October 2011
Line 18:		Line 18:
	##About 1/2 to 3/4 volume of the Overlap PCR reaction should be equimolar amounts of purified fragments.		##About 1/2 to 3/4 volume of the Overlap PCR reaction should be equimolar amounts of purified fragments.
	##Do not use Phusion polymerase. Try Pfu Turbo.		##Do not use Phusion polymerase. Try Pfu Turbo.
-	##DO not add any primers, the templates will prime ~~eachother~~	+	##Do not add any primers; the templates will prime each-other.
	##Run 15 PCR cycles without primers.		##Run 15 PCR cycles without primers.
	##Use an annealing temp of 60°C.		##Use an annealing temp of 60°C.
Line 31:		Line 31:
	##Select		##Select
	##Sequence		##Sequence
		+
	==Notes==		==Notes==
	*This protocol works best for assembling parts parts greater than 100bp. For making smaller parts see [[DNA Synthesis from Oligos]].		*This protocol works best for assembling parts parts greater than 100bp. For making smaller parts see [[DNA Synthesis from Oligos]].

Michael A. Speer at 22:06, 14 September 2011

Michael A. Speer — Wed, 14 Sep 2011 22:06:35 GMT

←Older revision		Revision as of 22:06, 14 September 2011
Line 1:		Line 1:
-	~~{{back~~ to protocols}}	+	Back to [[protocols]]
		+	[[Image:SOEing.PNG\|400px\|center]]
		+
	== Overview ==		== Overview ==
-	~~[[Image:SOEing.PNG\|400px\|right]]~~
	Create long DNA fragments from shorter ones. This method is also called "Splicing by Overlap Extension" or SOEing.		Create long DNA fragments from shorter ones. This method is also called "Splicing by Overlap Extension" or SOEing.

Michael A. Speer at 22:05, 14 September 2011

Michael A. Speer — Wed, 14 Sep 2011 22:05:47 GMT

←Older revision		Revision as of 22:05, 14 September 2011
Line 30:		Line 30:
	##Select		##Select
	##Sequence		##Sequence
		+	==Notes==
		+	*This protocol works best for assembling parts parts greater than 100bp. For making smaller parts see [[DNA Synthesis from Oligos]].

	[[Category:Protocol]]		[[Category:Protocol]]
	[[Category:DNA]]		[[Category:DNA]]
	[[Category:In vitro]]		[[Category:In vitro]]

Michael A. Speer: /* Overview */

Michael A. Speer — Wed, 14 Sep 2011 22:03:48 GMT

Overview

←Older revision		Revision as of 22:03, 14 September 2011
Line 1:		Line 1:
	{{back to protocols}}		{{back to protocols}}
	== Overview ==		== Overview ==
-	[[Image:SOEing.PNG~~\|right~~\|400px]]	+	[[Image:SOEing.PNG\|400px\|right]]
	Create long DNA fragments from shorter ones. This method is also called "Splicing by Overlap Extension" or SOEing.		Create long DNA fragments from shorter ones. This method is also called "Splicing by Overlap Extension" or SOEing.

Michael A. Speer: /* Overview */

Michael A. Speer — Wed, 14 Sep 2011 22:03:24 GMT

Overview

←Older revision		Revision as of 22:03, 14 September 2011
Line 1:		Line 1:
	{{back to protocols}}		{{back to protocols}}
	== Overview ==		== Overview ==
		+	[[Image:SOEing.PNG\|right\|400px]]
	Create long DNA fragments from shorter ones. This method is also called "Splicing by Overlap Extension" or SOEing.		Create long DNA fragments from shorter ones. This method is also called "Splicing by Overlap Extension" or SOEing.

Michael A. Speer: /* Procedure */

Michael A. Speer — Wed, 14 Sep 2011 21:30:10 GMT

Procedure

←Older revision		Revision as of 21:30, 14 September 2011
Line 17:		Line 17:
	##Do not use Phusion polymerase. Try Pfu Turbo.		##Do not use Phusion polymerase. Try Pfu Turbo.
	##DO not add any primers, the templates will prime eachother		##DO not add any primers, the templates will prime eachother
-	##Run 15 PCR cycles without primers.	+	##Run 15 PCR cycles without primers.
		+	##Use an annealing temp of 60°C.
	#"'''Purification PCR'''" Add end primers to the Overlap PCR reaction:		#"'''Purification PCR'''" Add end primers to the Overlap PCR reaction:
	##Continue cycling for another 15-20 rounds.		##Continue cycling for another 15-20 rounds.
		+	##Use an annealing temp of 72°C
	#Gel extract the correct size fragment.		#Gel extract the correct size fragment.
	#Clone into the desired vector.		#Clone into the desired vector.

Michael A. Speer: /* Procedure */

Michael A. Speer — Wed, 14 Sep 2011 21:24:08 GMT

Procedure

Michael A. Speer: /* Procedure */

Michael A. Speer — Wed, 14 Sep 2011 21:14:18 GMT

Procedure

←Older revision		Revision as of 21:14, 14 September 2011
Line 9:		Line 9:
	##These primers will each have a 60°C Tm with one part and a 60°C Tm with the other part.		##These primers will each have a 60°C Tm with one part and a 60°C Tm with the other part.
	##The "end primers" will not have any complements and will likely only have restriction sites.		##The "end primers" will not have any complements and will likely only have restriction sites.
-	#PCR amplify the necessary fragments separately:	+	#PCR amplify the necessary fragments separately "'''Extension PCR'''":
	##Use a proofreading polymerase enzyme.		##Use a proofreading polymerase enzyme.
	##Use an annealing temp of 60°C.		##Use an annealing temp of 60°C.
-	#Clean up ~~or gel extract~~ the ~~correct size band~~.	+	#Clean up the product using a DNA column.
-	#Use cleaned up fragments as template and primers in a PCR reaction.	+	#Use cleaned up fragments as template and primers in a PCR reaction (This is the "'''Overlap PCR'''".
-	##~~about~~ 1/2 to volume of the ~~extension~~ reaction should be equimolar amounts of purified fragments.	+	##About 1/2 to 3/4 volume of the Overlap PCR reaction should be equimolar amounts of purified fragments.
	##Do not use Phusion polymerase. Try Pfu Turbo.		##Do not use Phusion polymerase. Try Pfu Turbo.
	##Run 15 PCR cycles without primers. (Template extension step)		##Run 15 PCR cycles without primers. (Template extension step)

Michael A. Speer: /* Procedure */

Michael A. Speer — Wed, 14 Sep 2011 20:40:13 GMT

Procedure

←Older revision		Revision as of 20:40, 14 September 2011
Line 7:		Line 7:
	##These primers are like bridges between the two parts you want to assemble together.		##These primers are like bridges between the two parts you want to assemble together.
	##You will order two primers which are complements of one another.		##You will order two primers which are complements of one another.
-	##These primers will each have a 60°C Tm with one part and a 60°C Tm with the other part.	+	##These primers will each have a 60°C Tm with one part and a 60°C Tm with the other part.
		+	##The "end primers" will not have any complements and will likely only have restriction sites.
	#PCR amplify the necessary fragments separately:		#PCR amplify the necessary fragments separately:
	##Use a proofreading polymerase enzyme.		##Use a proofreading polymerase enzyme.

←Older revision		Revision as of 21:24, 14 September 2011
Line 9:		Line 9:
	##These primers will each have a 60°C Tm with one part and a 60°C Tm with the other part.		##These primers will each have a 60°C Tm with one part and a 60°C Tm with the other part.
	##The "end primers" will not have any complements and will likely only have restriction sites.		##The "end primers" will not have any complements and will likely only have restriction sites.
-	#~~PCR amplify the necessary fragments separately~~ "'''Extension PCR'''":	+	#"'''Extension PCR'''" PCR amplify the necessary fragments separately
	##Use a proofreading polymerase enzyme.		##Use a proofreading polymerase enzyme.
	##Use an annealing temp of 60°C.		##Use an annealing temp of 60°C.
	#Clean up the product using a DNA column.		#Clean up the product using a DNA column.
-	#~~Use cleaned up fragments as template and primers in a PCR reaction (This is the~~ "'''Overlap PCR'''".	+	#"'''Overlap PCR'''" Use cleaned up fragments as template in a PCR reaction:
	##About 1/2 to 3/4 volume of the Overlap PCR reaction should be equimolar amounts of purified fragments.		##About 1/2 to 3/4 volume of the Overlap PCR reaction should be equimolar amounts of purified fragments.
	##Do not use Phusion polymerase. Try Pfu Turbo.		##Do not use Phusion polymerase. Try Pfu Turbo.
-	##Run 15 PCR cycles without primers. ~~(Template extension step)~~	+	##DO not add any primers, the templates will prime eachother
-	#Add end primers	+	##Run 15 PCR cycles without primers.
		+	#"'''Purification PCR'''" Add end primers to the Overlap PCR reaction:
	##Continue cycling for another 15-20 rounds.		##Continue cycling for another 15-20 rounds.
	#Gel extract the correct size fragment.		#Gel extract the correct size fragment.