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High fidelity PCR trouble shooting

high fidelity pcr

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#1 uday

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Posted 23 May 2013 - 06:59 PM

I want to amplify a 1.8kb gene using error prone PCR for subsequent cloning purpose. My primers works well with normal tag polymerase (Fermentas Dreamtaq Green PCR master mix) and gave me the desired product. However, when I use the same primers and template with high fidelity tag polymerase (Platinum Tag DNA polymerase high fidelity - Invitrogen), I can't get any amplified DNA. What was the wrong? what would need to be changed? I am bit confused :(

#2 Curlis

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Posted 23 May 2013 - 09:44 PM

I want to amplify a 1.8kb gene using error prone PCR for subsequent cloning purpose. My primers works well with normal tag polymerase (Fermentas Dreamtaq Green PCR master mix) and gave me the desired product. However, when I use the same primers and template with high fidelity tag polymerase (Platinum Tag DNA polymerase high fidelity - Invitrogen), I can't get any amplified DNA. What was the wrong? what would need to be changed? I am bit confused Posted Image


Hi Uday,

Because of high fidelity, might be this enzyme is slow. You can try to use mix of Taq and this high fidelity enzyme. Mix them in different ratio (1:1, 2:1). I hope this will work.

#3 uday

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Posted 25 May 2013 - 06:45 PM

But will I be able to achieve my main aim, i.e., PCR product with minimum errors with the mixture of enzyme?





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