Dear Colleagues,
I have the following problem: I’m trying to amplify a cassette with a resistance gene (about 2 kb) from the yeast genome, so that I can then insert it into a plasmid. With Taq polymerase everything works. With proofread polymerase (Tersus) there is no PCR product. Everything works with the positive control (with ITS primers, the product is about 800 bp). I increased the elongation time to 4 minutes, increased the primary denaturation at 95 to 3 minutes. There is no the product. I would be very grateful for any advice.
Possibly your template has pcr inhibitors which may have a greater effect on your proofreading enzyme. Try amplifying less template dna (therefore less inhibitor). I assume that your ntp mix does not contain any modified ntps or utp which the proofreading enzyme would fail to incorporate
Paul Rutland
Dear Paul, thank you very much for your advice. I diluted template DNA 5 and 10 times, I will try to dilute it even more. We use regular NTP mix.
If this does not work then you could try a mixture of Taq and proofreading polymerase to get the best of both reactions....a product from the taq and proofread from the other enzyme. I assume that your no template control using the taq only is negative so that you can discount contamination of the taq reagents from previous amplifications
Paul Rutland
Dear Paul, very interesting advice, thank you. But will the proofreading effect be significant? If, for example, Taq completes an unfinished Tersus chain, and the Tersus chains are short, then the error level will be closer to Taq.
I think that the error rates are usually higher near the primers possibly due to extension by the enzyme at the annealing temp of the primer which may not be optimal for fidelity so it is possible that any benefit shows better on long pcr products Igor Mazheika
Hello Igor.
I faced the same issue in viral amplification using two PCR Master Mixes one for general detection and the other for Proofreading. I highly agree with Paul in PCR inhibition.
one more issue that seemed to cause this problem is the difference between Tm for the same primers. Proofreading enzymes tend to have greater TA than other taq enzymes. For my Proofreading mastermix, it requires 6-9 higher degrees than the taq polymerase for the same primers and same template. Try to contact the manufacturer of the proofreading enzyme if it has special calculation for TA.
best of luck
Ibrahim H M Abualghusein
Dear Ibrahim, thanks for the advice. I tried different annealing temperatures on a gradient cycler - with no result.
I was finally able to amplify the desired sequence using Tersus. Ibrahim was right, an increased annealing temperature is needed here. But not only that, I got a product using a combination of conditions (I tried high annealing temperatures and before): a lot of DNA template, few primers (
Good news Igor Mazheika well done and thanks for letting us know thesolution
Happy to hear that you managed to solve your issue. Best of Luck
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