Question

PCR improvement with Phusion Hi Fi taq polymerase

Hello, I'm doing a PCR with Phusion taq polymnerase with a cDNA (from RT) of 5 kb. I get a lot of non specific bands in addition of the desired one. I tried to do a temperature gradient but it didn't improve. It got better using GC buffer, but only a little bit. Is there any enhancer that works with this enzyme?
Sep 12, 2012

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All Answers (5)

  • Norma Suely de Lima Freitas · Federal University of Amazonas
    I guess you will have to improve your protocol trying optimizing the annealing temperatures and revise the design of your primers. It is fundamental to know exactly the extent and the conserved region of your primers that you want to amplify. Continue using the HF polymerase enzyme. Good luck!
    Sep 13, 2012
  • Mark Stead · Albert Einstein College of Medicine
    I've had very little success with Phusion even after annealing temperature gradients and multiple primer sets. I'm now using KOD hotstart (Novagen), and KOD Xtreme for high-GC content templates. I've supervised the cloning of over 8,000 genes using these enzymes and can attest to both their robustness and high fidelity rate.
    Sep 13, 2012
  • Kenzi Saito · Baylor College of Medicine
    I recommend you use Toyobo's KOD plus. I have used this enzyme in a variety of applications like DIG PCR labeling, genotyping, subcloning, and so on. One reason is because the company provides detailed information about options that can be applied when you face some problem in PCR.
    Sep 14, 2012
  • Kejun Guo · University of Colorado Denver
    Was the cDNA generated from specific primers or random hexamer? If the later, it's not surprising to have multiple bands at all. I have done countless cloning using Phusion with success. IMHO, it's most like not the problem with Phusion taq.
    Oct 3, 2012

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