I am detecting mutations in virus genome, using PCR for 1kb followed by Sanger sequencing. GoTaq (Promega) works greatlly, but PCR with Pfu Polymerase gives a small amount of amplicon. I followed all the instructions of elongation time, anneling temperature ...
Which enzyme should I use instead of Pfu Polymerase, that is a high fidelity (and not sooo expensive)??
If you are directly sequencing the PCR product (and not cloning it first) then it doesn't matter which polymerase you use. You can go ahead and use regular Taq or any variant thereof. You only need to use high fidelity polymerase if you are generating a single clone, but if you are sequencing the population of molecules following amplification, then the average sequence of the population will be correct. So again, if you are sequencing the PCR product directly, use any version of Taq. If you are cloning first and sequencing a single clone, the you should use a high fidelity polymerase of your choosing, I've always had good results with the mixed polymerases with Taq plus some high fidelity polymerase for error correction.
we had good results using both Gotaq hotstart and Qiagen hotstart polymerase. you can also try AmpliTaq Gold® 360 DNA Polymerase .
But because your DNA is long you may want to increase the number of cycles and increase the extension time.
If you are directly sequencing the PCR product (and not cloning it first) then it doesn't matter which polymerase you use. You can go ahead and use regular Taq or any variant thereof. You only need to use high fidelity polymerase if you are generating a single clone, but if you are sequencing the population of molecules following amplification, then the average sequence of the population will be correct. So again, if you are sequencing the PCR product directly, use any version of Taq. If you are cloning first and sequencing a single clone, the you should use a high fidelity polymerase of your choosing, I've always had good results with the mixed polymerases with Taq plus some high fidelity polymerase for error correction.
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