Hi all,
When I am conducting PCR in order to amplify a certain DNA segment for subsequent Sanger sequencing, would using the conventional DreamTaq Green Mix from Thermo Fisher work fine? Or should I opt for higher fidelity by using the Phusion High-Fidelity Master Mix, also from Thermo Fisher, instead? Thanks.
Hey Frank,
what is the reason for sending it for sequencing? If you (only) want to check if you have the correct Product or check for e.g. gene disruptions (as we do it), normal Taq amplification is fine. It also depends on the lenght of your PCR product. For small amplicons (below 500 bp) Taq is definitely sufficient.
regards Chris
I agree with the above answer
To elaborate:
Unless your template is complex, long, or hard to amplify, just use normal Taq. The error rate for commercial polymerases is negligible for sending your products for Sanger. If you are planning to clone and then sequence, then I'd also use the high-fidelity. But for normal PCR followed by Sanger, don't waste your money on the expensive enzyme.
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