Hello,
I want to do some cloning and for that I'll need to amplify the whole plasmid (~13 kbp), so I'm looking for DNA Polymerase which would have low mutation rate. We have Takara's PrimeSTAR GXL DNA Polymerase in our lab. In their FAQ, they claim it has superior fidelity, higher that Pfu or Phusion Q4/p3).
https://www.takarabio.com/documents/Frequently%20Asked%20Questions/PCRPolymerases_FAQ.pdf
Everybody seems to be excited about NEB's Q5 DNA Polymerase. There is a paper, which claims that Q5's fidelity is superior
Article Examining Sources of Error in PCR by Single-Molecule Sequencing
but the authors are NEB's employees.
In that paper, PS GXL DNA Polymerase is worse than Pfu and Phusion, so...
Which DNA Polymerase would you recommend? Thank you
I would not worry too much about it so long as your using one of the high fidelity polymerases. Slight variations in experimental conditions can impact the "reported" mutation rate, so the reported differences may not be particularly relevant. Also you really only need to worry about mutations in your gene and presumably you can sequence and confirm it is correct afterwards. You don't really care too much about silent mutations in the plasmid, and any mutations in the plasmid that impact replication or resistance will be lost because those plasmids won't transform. So you have a selection for a functional plasmid.
Dear Tomas,
in my Lab we use Takara's PrimeSTAR GXL DNA Polymerase only for difficult to amplify template (with high GC content), but routinely (mainly for cloning) we utilize Phusion polymerase, with no problems to date.
Good luck!
Chris
For amplification of a big vector >10 kb, it's better to go with Phusion or NEB Q5. In my experience, Q5 Pol gave even better results and thicker band. Although, I'd do gel purification after either of these for better specificity.
Hello, thanks for your inputs everyone.
Michael J. Benedik those are plasmids for in planta expression, so there's plenty of stuff (like T-DNA borders, selection for plant, which are not selected for (against?) in bacteria, so I'll need to check it afterwards anyway. But OK, I won't worry about it that much.
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