McClean: Takara PrimeStar PCR

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(New page: <!-- COPY EVERYHING BELOW HERE TO START YOUR OWN PROTOCOL! --> ==Overview== Takara PrimeSTAR HS DNA Polymerase is a high fidelity PCR enzyme. Use this enzyme and protocol for amplifying...)
Current revision (20:02, 29 June 2012) (view source)
(Overview)
 
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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==  
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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
+
*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 1min
+
**72.0°C for 2min 30sec
-
4°C forever
+
*72.0°C for 1min
 +
*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==
<!-- 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.
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*[[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==

Current revision


Contents

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
  • Repeat the following three lines 30 times:
    • 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 '''*~~~~''': to the beginning of your tip.

  • 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

Takara

Contact

or instead, discuss this protocol.

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