Replacing Taq with Pfu in PCR reaction

[ssDNA]

Hi guys. I was wondering if anyone have any experience replacing Taq with Pfu with PCR reactions? Under what circumstances would they work and under what circumstances would this not work?

[Putra Taufik]

i did use pfu( Fermentas) to amplify a band after i optimized the condition using taq. but the result is not good. multiple bands were obtained although only single band was produced by taq. pfu not works well for my case, i think proble due to the pfu.because i try a few enzyme to amplify the same product(2.5kb). pfu took me 4-5 hours, and the yield very low, not suitable for cloning.
now i m looking for other enzyme with high fidelity and proofreading activity to amplify the product. hope someone can tell me what enzyme is good for my case, thank you.

[jonoave]

It depends. Pfu is more selective and difficult work with. I use regular Taq to do a quick screening whether the PCR reaction works (template/primer design etc) before I switch to Pfu.

[d.alhajturki]

hi, I'm using Pfu just in case of isolation , cloning and sequencing to make sure from sequence fidelity .. but PCR reaction must be optimised (DNA tempalt and primers..etc) and in some cases may be u have to use PCR enhancer if there is no product or low yield... in PCR programme the extension tm depends on the size of the amplified fragment.. 1 min per Kb decreasing cycles number to minimize non-specific products

[alkasethi]

Hi i have used pfu for amplifying the high GC rich regions and mitochondrial genome. It worked well for me , initially i did get a few nonspecific bands but later they diappeared.