Corum:Whole Plasmid PCR - Revision history

Sean P Corum: /* Materials */

Sean P Corum — Thu, 13 Dec 2012 18:10:51 GMT

Materials

←Older revision		Revision as of 18:10, 13 December 2012
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	* 37 μL H<sub>2</sub>O		* 37 μL H<sub>2</sub>O
	* 5 μL 10X PFU Ultra PCR buffer		* 5 μL 10X PFU Ultra PCR buffer
-	* 5 μL 10 μM sense/antisense primer mix (0.5 μM final)	+	* 5 μL 10 μM sense/antisense primer mix (0.5 μM final, each)
	* 1 μL 12.5 mM (each) dNTP mix (0.25 mM final)		* 1 μL 12.5 mM (each) dNTP mix (0.25 mM final)
	* 1 μL 5 nM plasmid template (0.1 nM final)		* 1 μL 5 nM plasmid template (0.1 nM final)

Sean P Corum: /* Overview */

Sean P Corum — Thu, 13 Dec 2012 17:53:50 GMT

Overview

←Older revision		Revision as of 17:53, 13 December 2012
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	==Overview==		==Overview==
-	Whole plasmid polymerase chain reaction (WP-PCR) protocol using [http://www.genomics.agilent.com/CollectionSubpage.aspx?PageType=Product&SubPageType=ProductDetail&PageID=1095 PfuUltra II fusion HS DNA polymerase]. It is imperative to use 5'-phosphorylated primers if the nicked DNA is going to be repaired downstream with ligase. PCR should be limited to templates of about 5 kb, but the protocol could probably be pushed up to 10 kb. See the PfuUltra II fusion manual.	+	Whole plasmid polymerase chain reaction (WP-PCR) protocol using [http://www.genomics.agilent.com/CollectionSubpage.aspx?PageType=Product&SubPageType=ProductDetail&PageID=1095 PfuUltra II fusion HS DNA polymerase]. It is imperative to use 5'-phosphorylated primers if the nicked DNA is going to be repaired downstream with ligase. PCR should be limited to templates of about 5 kb, but the protocol could probably be pushed up to 10 kb. See the PfuUltra II fusion manual. Primers should be sense-antisense pairs. Optimal amplification occurs with 30-35 b primers at 0.5 μM final concentration. At higher concentrations, the primers bind to each other, inhibiting amplification. For longer primers, the annealing temperature will need to be adjusted from the one mentioned in this protocol.

	==Materials==		==Materials==

Sean P Corum at 17:50, 13 December 2012

Sean P Corum — Thu, 13 Dec 2012 17:50:20 GMT

←Older revision		Revision as of 17:50, 13 December 2012
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	==Overview==		==Overview==
-	Whole plasmid polymerase chain reaction (WP-PCR) protocol using [http://www.genomics.agilent.com/CollectionSubpage.aspx?PageType=Product&SubPageType=ProductDetail&PageID=1095 PfuUltra II fusion HS DNA polymerase]~~. [http://www.genomics.agilent.com/CollectionSubpage.aspx?PageType=Product&SubPageType=ProductDetail&PageID=1147 PFU ligase] is used in parallel to repair the nicked plasmid products as PCR proceeds~~. It is imperative to use 5'-phosphorylated primers ~~for~~ the ligase to be ~~able~~ to ~~repair~~ the ~~nicks~~.	+	Whole plasmid polymerase chain reaction (WP-PCR) protocol using [http://www.genomics.agilent.com/CollectionSubpage.aspx?PageType=Product&SubPageType=ProductDetail&PageID=1095 PfuUltra II fusion HS DNA polymerase]. It is imperative to use 5'-phosphorylated primers if the nicked DNA is going to be repaired downstream with ligase. PCR should be limited to templates of about 5 kb, but the protocol could probably be pushed up to 10 kb. See the PfuUltra II fusion manual.

	==Materials==		==Materials==
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	For a 50 μL WP-PCR reaction:		For a 50 μL WP-PCR reaction:

-	* 36 μL H<sub>2</sub>O	+	* 37 μL H<sub>2</sub>O
	* 5 μL 10X PFU Ultra PCR buffer		* 5 μL 10X PFU Ultra PCR buffer
-	* 5 μL ~~100~~ μM sense/antisense primer mix (10 μM final)	+	* 5 μL 10 μM sense/antisense primer mix (0.5 μM final)
	* 1 μL 12.5 mM (each) dNTP mix (0.25 mM final)		* 1 μL 12.5 mM (each) dNTP mix (0.25 mM final)
	* 1 μL 5 nM plasmid template (0.1 nM final)		* 1 μL 5 nM plasmid template (0.1 nM final)
	* 1 μL [http://www.genomics.agilent.com/CollectionSubpage.aspx?PageType=Product&SubPageType=ProductDetail&PageID=1095 PfuUltra II fusion HS DNA polymerase]		* 1 μL [http://www.genomics.agilent.com/CollectionSubpage.aspx?PageType=Product&SubPageType=ProductDetail&PageID=1095 PfuUltra II fusion HS DNA polymerase]
-	* 1 μL [http://www.genomics.agilent.com/CollectionSubpage.aspx?PageType=Product&SubPageType=ProductDetail&PageID=1147 PFU ligase]

	==Procedure==		==Procedure==
-	# In a PCR tube, ~~mix~~ the components on ice in the order they are listed above. Mix gently and spin.	+	# In a PCR tube, add the components on ice in the order they are listed above. Mix gently and spin.
	# Perform the following thermocycling program:		# Perform the following thermocycling program:
	## Initial melting: 95 °C 2 min		## Initial melting: 95 °C 2 min
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	## Annealing: T<sub>a</sub> °C 20 s, where T<sub>a</sub> = T<sub>m</sub> - 5 °C		## Annealing: T<sub>a</sub> °C 20 s, where T<sub>a</sub> = T<sub>m</sub> - 5 °C
	## Elongation: 72 °C 2 min / kb template		## Elongation: 72 °C 2 min / kb template
-	~~## Ligation: 55 °C 30 s (time?)~~
	## Repeat steps 2-4 a total of 30 times		## Repeat steps 2-4 a total of 30 times
	## Final elongation: 72 °C 30 min		## Final elongation: 72 °C 30 min
-	~~## Final ligation: 55 °C 1 hr~~	+	## 12-16 °C hold
-	## 12 °C hold	+
	# Verify product with [http://openwetware.org/wiki/Corum:Gel_Electrophoresis gel electrophoresis].		# Verify product with [http://openwetware.org/wiki/Corum:Gel_Electrophoresis gel electrophoresis].
	# Quantify product with [http://openwetware.org/wiki/Corum:DNA_Quantification quantifluore DNA quantification].		# Quantify product with [http://openwetware.org/wiki/Corum:DNA_Quantification quantifluore DNA quantification].
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	==Contact==		==Contact==
	* [http://openwetware.org/wiki/User:Sean_P_Corum Sean P Corum]		* [http://openwetware.org/wiki/User:Sean_P_Corum Sean P Corum]
-	*'''SC 18:46, ~~23 July~~ 2012 (~~EDT~~)''':	+	*'''SC 12:50, 13 December 2012 (EST)''':

	or instead, [[Talk:{{PAGENAME}}\|discuss this protocol]].		or instead, [[Talk:{{PAGENAME}}\|discuss this protocol]].

Sean P Corum: /* Materials */

Sean P Corum — Tue, 04 Sep 2012 19:53:27 GMT

Materials

←Older revision		Revision as of 19:53, 4 September 2012
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	==Materials==		==Materials==

-	For a 50 μL ~~whole plasmid PCR~~ PCR reaction:	+	For a 50 μL WP-PCR reaction:

	* 36 μL H<sub>2</sub>O		* 36 μL H<sub>2</sub>O

Sean P Corum: /* Procedure */

Sean P Corum — Sun, 26 Aug 2012 03:19:25 GMT

Procedure

←Older revision		Revision as of 03:19, 26 August 2012
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	## Initial melting: 95 °C 2 min		## Initial melting: 95 °C 2 min
	## Melting: 95 °C 20 s		## Melting: 95 °C 20 s
-	## Annealing: T<sub>a</sub> °C = 20 s, where T<sub>a</sub> = T<sub>m</sub> - 5 °C	+	## Annealing: T<sub>a</sub> °C 20 s, where T<sub>a</sub> = T<sub>m</sub> - 5 °C
	## Elongation: 72 °C 2 min / kb template		## Elongation: 72 °C 2 min / kb template
	## Ligation: 55 °C 30 s (time?)		## Ligation: 55 °C 30 s (time?)

Sean P Corum: /* Procedure */

Sean P Corum — Sun, 26 Aug 2012 03:19:04 GMT

Procedure

←Older revision		Revision as of 03:19, 26 August 2012
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	## Elongation: 72 °C 2 min / kb template		## Elongation: 72 °C 2 min / kb template
	## Ligation: 55 °C 30 s (time?)		## Ligation: 55 °C 30 s (time?)
-	## Repeat steps 2-4 a total of ~~12-20~~ times ~~(20 is standard).~~	+	## Repeat steps 2-4 a total of 30 times
	## Final elongation: 72 °C 30 min		## Final elongation: 72 °C 30 min
	## Final ligation: 55 °C 1 hr		## Final ligation: 55 °C 1 hr

Sean P Corum at 03:18, 26 August 2012

Sean P Corum — Sun, 26 Aug 2012 03:18:34 GMT

←Older revision		Revision as of 03:18, 26 August 2012
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	For a 50 μL whole plasmid PCR PCR reaction:		For a 50 μL whole plasmid PCR PCR reaction:

-	* 37 μL H<sub>2</sub>O	+	* 36 μL H<sub>2</sub>O
	* 5 μL 10X PFU Ultra PCR buffer		* 5 μL 10X PFU Ultra PCR buffer
	* 5 μL 100 μM sense/antisense primer mix (10 μM final)		* 5 μL 100 μM sense/antisense primer mix (10 μM final)
Line 12:		Line 12:
	* 1 μL 5 nM plasmid template (0.1 nM final)		* 1 μL 5 nM plasmid template (0.1 nM final)
	* 1 μL [http://www.genomics.agilent.com/CollectionSubpage.aspx?PageType=Product&SubPageType=ProductDetail&PageID=1095 PfuUltra II fusion HS DNA polymerase]		* 1 μL [http://www.genomics.agilent.com/CollectionSubpage.aspx?PageType=Product&SubPageType=ProductDetail&PageID=1095 PfuUltra II fusion HS DNA polymerase]
		+	* 1 μL [http://www.genomics.agilent.com/CollectionSubpage.aspx?PageType=Product&SubPageType=ProductDetail&PageID=1147 PFU ligase]

	==Procedure==		==Procedure==

Sean P Corum at 03:17, 26 August 2012

Sean P Corum — Sun, 26 Aug 2012 03:17:47 GMT

←Older revision		Revision as of 03:17, 26 August 2012
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	* 1 μL 12.5 mM (each) dNTP mix (0.25 mM final)		* 1 μL 12.5 mM (each) dNTP mix (0.25 mM final)
	* 1 μL 5 nM plasmid template (0.1 nM final)		* 1 μL 5 nM plasmid template (0.1 nM final)
-	* 1 μL ~~PFU Ultra~~ II ~~high-fidelity~~ DNA ~~polyermerase~~	+	* 1 μL [http://www.genomics.agilent.com/CollectionSubpage.aspx?PageType=Product&SubPageType=ProductDetail&PageID=1095 PfuUltra II fusion HS DNA polymerase]

	==Procedure==		==Procedure==
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	## Annealing: T<sub>a</sub> °C = 20 s, where T<sub>a</sub> = T<sub>m</sub> - 5 °C		## Annealing: T<sub>a</sub> °C = 20 s, where T<sub>a</sub> = T<sub>m</sub> - 5 °C
	## Elongation: 72 °C 2 min / kb template		## Elongation: 72 °C 2 min / kb template
		+	## Ligation: 55 °C 30 s (time?)
	## Repeat steps 2-4 a total of 12-20 times (20 is standard).		## Repeat steps 2-4 a total of 12-20 times (20 is standard).
	## Final elongation: 72 °C 30 min		## Final elongation: 72 °C 30 min
		+	## Final ligation: 55 °C 1 hr
	## 12 °C hold		## 12 °C hold
	# Verify product with [http://openwetware.org/wiki/Corum:Gel_Electrophoresis gel electrophoresis].		# Verify product with [http://openwetware.org/wiki/Corum:Gel_Electrophoresis gel electrophoresis].

Sean P Corum at 03:15, 26 August 2012

Sean P Corum — Sun, 26 Aug 2012 03:15:39 GMT

←Older revision		Revision as of 03:15, 26 August 2012
Line 1:		Line 1:
	==Overview==		==Overview==
-	WP-PCR protocol using [http://www.genomics.agilent.com/CollectionSubpage.aspx?PageType=Product&SubPageType=ProductDetail&PageID=1095 PfuUltra II fusion HS DNA polymerase]. [http://www.genomics.agilent.com/CollectionSubpage.aspx?PageType=Product&SubPageType=ProductDetail&PageID=1147 PFU ligase] is used in parallel to repair the nicked plasmid products as PCR proceeds. It is imperative to use 5'-phosphorylated primers for the ligase to be able to repair the nicks.	+	Whole plasmid polymerase chain reaction (WP-PCR) protocol using [http://www.genomics.agilent.com/CollectionSubpage.aspx?PageType=Product&SubPageType=ProductDetail&PageID=1095 PfuUltra II fusion HS DNA polymerase]. [http://www.genomics.agilent.com/CollectionSubpage.aspx?PageType=Product&SubPageType=ProductDetail&PageID=1147 PFU ligase] is used in parallel to repair the nicked plasmid products as PCR proceeds. It is imperative to use 5'-phosphorylated primers for the ligase to be able to repair the nicks.

	==Materials==		==Materials==

Sean P Corum: /* Overview */

Sean P Corum — Sun, 26 Aug 2012 03:14:58 GMT

Overview

←Older revision		Revision as of 03:14, 26 August 2012
Line 1:		Line 1:
	==Overview==		==Overview==
-	~~Whole plasmid~~ PCR protocol using ~~PFU Ultra~~ DNA polymerase. PFU ligase is used in ~~the reaction~~ to repair the nicked plasmid products as PCR proceeds. It is imperative to use 5'-phosphorylated primers for the ligase to repair the nicks.	+	WP-PCR protocol using [http://www.genomics.agilent.com/CollectionSubpage.aspx?PageType=Product&SubPageType=ProductDetail&PageID=1095 PfuUltra II fusion HS DNA polymerase]. [http://www.genomics.agilent.com/CollectionSubpage.aspx?PageType=Product&SubPageType=ProductDetail&PageID=1147 PFU ligase] is used in parallel to repair the nicked plasmid products as PCR proceeds. It is imperative to use 5'-phosphorylated primers for the ligase to be able to repair the nicks.

	==Materials==		==Materials==