Pfu polymerase problem

[prot]

Hi. I am running my pcr for two of my genes of interest. I have successfully amplified the target gene using normal Taq polymerase. For higher fidelity purpose, I try to run the PCR using Pfu polymerase with the same condition as the previous run. However, no band was observed on the agarose gel. What might be the problem? I tried to follow the manufacturer instruction, increase the extension time, increase polymerase conc and use recommended conc of the reagent. I still fail to amplify my gene. Can anyone advice me?

[jonoave]

In my experience, there are some stuff they don't tell you bout Pfu. In my experience:

1. Pfu has a lower Tm range that normal Taq - Using the same Tm as you did for Taq won't necessarily give you the same product, or any at all. What I do is first of all to test the presence of a product using Taq (because Pfu is more expensive). Then after that, I use Pfu starting with the lowest range in Taq.

Eg: I run with Taq at 58, 60, 62, 64. At Tm 60-64, a desired band size is seen. Then I rerun with Pfu at 54, 56, 58, 60 (the highest range being the lowest range achievable in Taq).

2. Pfu is less efficient than Taq - Ok, they tell you this. But not really what this means. This means that you generally need a higher amount of Pfu than Taq. Especially if you are adding inhibitors/additives eg glycerol/betaine, you need to increase the amount of Pfu you're using. Eg: Using 1U of Taq is more than sufficient for me, even with inhibitors. But with Pfu, I use 1.25 U for normal reactions, and up to 2 U with inhibitors.

Or if for some reason your amplification is less than efficient (the band intensity isn't very bright in Taq-PCR run), try running a nested PCR or a second reamplification. For me,