McClean: Takara PrimeStar PCR - Revision history

Megan N McClean: /* Overview */

Megan N McClean — Sat, 30 Jun 2012 00:02:31 GMT

Overview

←Older revision		Revision as of 00:02, 30 June 2012
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	==Overview==		==Overview==
	Takara PrimeSTAR HS DNA Polymerase is a high fidelity PCR enzyme. Use this enzyme and protocol for amplifying PCR products for transformation, cloning, etc (basically any application where you want high fidelity.) Don't use this for things like colony PCR where you don't care if the product has mutations (use the cheap stuff for colony PCR!)		Takara PrimeSTAR HS DNA Polymerase is a high fidelity PCR enzyme. Use this enzyme and protocol for amplifying PCR products for transformation, cloning, etc (basically any application where you want high fidelity.) Don't use this for things like colony PCR where you don't care if the product has mutations (use the cheap stuff for colony PCR!)
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	==Materials==		==Materials==

Megan N McClean: /* PCR Program */

Megan N McClean — Sat, 30 Jun 2012 00:02:04 GMT

PCR Program

←Older revision		Revision as of 00:02, 30 June 2012
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	Run PCR Program (labeled takarapcr on thermocycler)		Run PCR Program (labeled takarapcr on thermocycler)
	*94.0°C for 2 min		*94.0°C for 2 min
-	*98.0°C for 10 sec	+	*Repeat the following three lines 30 times:
-	*53.0°C for 15sec Repeat 30 times	+	**98.0°C for 10 sec
-	*72.0°C for 2min 30sec	+	**53.0°C for 15sec Repeat 30 times
		+	**72.0°C for 2min 30sec
	*72.0°C for 1min		*72.0°C for 1min
	*4°C forever		*4°C forever

Megan N McClean: /* Protocol */

Megan N McClean — Sat, 30 Jun 2012 00:01:29 GMT

Protocol

←Older revision		Revision as of 00:01, 30 June 2012
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	===PCR Program===		===PCR Program===
	Run PCR Program (labeled takarapcr on thermocycler)		Run PCR Program (labeled takarapcr on thermocycler)
-	94.0°C for 2 min	+	*94.0°C for 2 min
-	98.0°C for 10 sec	+	*98.0°C for 10 sec
-	53.0°C for 15sec Repeat 30 times	+	*53.0°C for 15sec Repeat 30 times
-	72.0°C for 2min 30sec	+	*72.0°C for 2min 30sec
-	72.0°C for 1min	+	*72.0°C for 1min
-	4°C forever	+	*4°C forever

	Adjust the extension time according to the expected length of your product (~1 min per kb).		Adjust the extension time according to the expected length of your product (~1 min per kb).
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	==Notes==		==Notes==

Megan N McClean: /* Notes */

Megan N McClean — Sat, 30 Jun 2012 00:01:10 GMT

Notes

←Older revision		Revision as of 00:01, 30 June 2012
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	<!-- Please paste this section "as is" into your protocol, and add notes to it if you have some!-->		<!-- Please paste this section "as is" into your protocol, and add notes to it if you have some!-->
	Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input! Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.		Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input! Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.
-	*[[User:Megan N McClean\|Megan N McClean]] 17:48, 29 June 2012 (EDT) As with all PCR reactions, it usually makes sense to make up a master mix (polymerase, DNTPs, and buffer) and then add primers and DNA individually, rather than trying to pipette minute quantities into each individual PCR tube. Use your own good judgement ~~(or add something~~ to ~~this protocol to clarify the~~ master ~~mix principle for other users!)~~	+	*[[User:Megan N McClean\|Megan N McClean]] 17:48, 29 June 2012 (EDT) As with all PCR reactions, it usually makes sense to make up a master mix (polymerase, DNTPs, and buffer) and then add primers and DNA individually, rather than trying to pipette minute quantities into each individual PCR tube. Use your own good judgement. If someone has time it might be nice/useful to write up an explanation of master mixes somewhere on our OWW site.

	==References==		==References==

Megan N McClean: New page: ==Overview== Takara PrimeSTAR HS DNA Polymerase is a high fidelity PCR enzyme. Use this enzyme and protocol for amplifying...

Megan N McClean — Sat, 30 Jun 2012 00:00:25 GMT

New page:  ==Overview== Takara PrimeSTAR HS DNA Polymerase is a high fidelity PCR enzyme. Use this enzyme and protocol for amplifying...

New page

==Overview==
Takara PrimeSTAR HS DNA Polymerase is a high fidelity PCR enzyme. Use this enzyme and protocol for amplifying PCR products for transformation, cloning, etc (basically any application where you want high fidelity.) Don't use this for things like colony PCR where you don't care if the product has mutations (use the cheap stuff for colony PCR!)

==Materials==
* 5X Takara Primestar Buffer
* DNTPS (2.5mM each)
* Takara Primestar Polymerase
These reagents (buffer, DNTPs, and polymerase) are supplied in kit R010A or R010B (which we currently order through Fisher)
* Primer 1 (10μM)
* Primer 2 (10μM)
* Plasmid DNA (20ng/μl concentration) (or other DNA source)
* Sterile Water

==Protocol==

===Reaction Mix===
For each 50μL reaction the reaction mix is as follows:
* 5X Takara Primestar Buffer 10μl
* DNTPS (2.5mM each) 4μl
* Primer 1 (10μM) 1μl
* Primer 2 (10μM) 1μl
* Plasmid DNA (20ng/μl concentration) 1μl
* Takara Primestar Polymerase 0.5μl
* Sterile Water 32.5μl

===PCR Program===
Run PCR Program (labeled takarapcr on thermocycler)
94.0°C for 2 min
98.0°C for 10 sec
53.0°C for 15sec Repeat 30 times
72.0°C for 2min 30sec
72.0°C for 1min
4°C forever

Adjust the extension time according to the expected length of your product (~1 min per kb).

==Notes==

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input! Please sign your name to your note by adding <font face="courier"><nowiki>'''*~~~~''':</nowiki></font> to the beginning of your tip.
*[[User:Megan N McClean|Megan N McClean]] 17:48, 29 June 2012 (EDT) As with all PCR reactions, it usually makes sense to make up a master mix (polymerase, DNTPs, and buffer) and then add primers and DNA individually, rather than trying to pipette minute quantities into each individual PCR tube. Use your own good judgement (or add something to this protocol to clarify the master mix principle for other users!)

==References==
[http://www.clontech.com/takara Takara]

==Contact==

*'''[[User:Megan N McClean|Megan N McClean]] 14:01, 20 July 2011 (EDT)'''

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


[[Category:Protocol]]