Pfu polymerase from promega - (Jul/11/2006 )
Does anybody here use pfu polymerase from promega for PCR amplification please? I used to use pfu turbo from stratagene for proof reading (95 degree for 2min initial denaturation and 30sec for each cycle, 5 degree below the primer Tm for annealing,72 degree extension for 1kb/min, and 10 min 72 degree extra extention)and everything works fine. However, after i changed enzyme to pfu polymerase from promega while all the pcr condition remained the same, nothing worked any more. Then i changed the extention time to 1kb/2 min (72 degree for extension temp) as it was recommended in the product sheet, and the annealing temperature was 5 degree below the Tm of the primers. Yet still nothing worked.
Has any body used this enzyme before and got it worked in the pcr? May I have some suggestions in optimising the enzyme please?
Thank you !!!!!!
I tried it and it's very well for RT PCR
for routine PCR I idvice you to use
GoTaq DNA polymerase (promega)
Taq polymerase (Invitrogen)
the salt concentration for each enzyme 1.5mM
Pfu Turbo is Pfu plus thermostable ArchaeMaxx™ polymerase-enhancing factor, one a flavoprotein, the other a dCTP deaminase (dUTPase). Together they make a complex which enhances Pfu.
According to Stratagene, "the enhanced performance of PfuTurbo DNA polymerase allows the use of shorter extension times, fewer PCR cycles, and lower DNA template concentrations than are required for Pfu DNA polymerase. The ArchaeMaxx factor improves the yield of products by overcoming dUTP poisoning, which is caused by dUTP accumulation during PCR through dCTP deamination.5 Once incorporated, dU-containing DNA inhibits Pfu and most archaeal proofreading DNA polymerases, such as Vent® and Deep Vent® DNA polymerases, limiting their efficiency. The ArchaeMaxx factor functions as a dUTPase, converting poisonous dUTP to harmless dUMP and inorganic pyrophosphate, resulting in improved overall PCR performance."
ArchaeMaxx is not available as a separate component.
To Najib: Thank you very much for the advice. According to your experience, perhaps I should try to use the cDNA library as the template rather than use the RT-PCR product as the template. However, the reason I use pfu polymerase is that i need its high-fidelity properties.
To tfitzwater: Thank you for your introduction in Pfu turbo.
P.S: is it ok to use the enzyme for introducing mutation in to my sequence using the pfu polymerase from promega?
You can use Pfu for site directed mutagenesis if that's what you want to do. Pfu and its variants/variant formulations (pfuturbo and pfuultra) are the most widely used for SDM. More info on www.stratagene.com (search for quickchange). Btw: you don't have to buy the kit, you can easily buy the components separately (also from different suppliers) to make it cheaper...