McClean: Takara PrimeStar 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!)
- 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
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
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).
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.
- Megan N McClean 14:01, 20 July 2011 (EDT)
or instead, discuss this protocol.