Hi all,
So I want to clone out a gene from a commercially obtained cDNA library. I was wondering if people have been successful using a nested primer approach for a gene of almost 6Kb? I have some unique restriction sites in the middle of the gene that I could paste together if need be. I'd like to design my nested primers to contain cloning restriction sites and would like some input on polymerases that would do the job. I was looking at a high fidelity polymerase from takara/clotech
http://www.clontech.com/US/Products/PCR/High_Fidelity_PCR/Advantage_HD_DNA_Polymerase?sitex=10020:22372:US
Thoughts?
Thanks.
Hi,
The Q5 (NEB) should do the job as well. It is a high fidelity polymerase with proof reading activity. This enzyme worked always very well in my hands for amplification of sequences of similar sizes. However, most available high fidelity polymerases will be suitable for this job.
What do you mean by 'nested primer approach'? Using one pair of primers should be enough. There is no need for multiple PCRs with nested primers. Additional amplification rounds will increase the risk of integrating errors.
Good luck with your PCR!
Agree with Boas about not using nested PCR. I have amplified around 10kb using Phusion High fidelity DNA polymerase (NEB or Thermofisher) successfully .
Agree with Damle. Phusion High fidelity DNA pol (NEB) works very well for inverse PCRs on plasmids. I believe, for 6kb amplification this would work just fine.
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