Just a quick question for the practising biologists on the forum. I've just started a research project and will be performing some nested PCR on a 4kb sequence that will be genotyped in a number of individuals (~50 initially). Does anyone know, off the top of their heads, of a high fidelity polymerase that is capable of performing long PCR's? I'm going to be testing some pre-designed primers and such, and all the other reagents and thermal profile are going to be tested empirically so they aren't an issue right now.

 

Cheers in advance.

From the top of my head, polymerases with proof reading capability and with with which I successfully amplified ~10 kb of high GC fragments (most are able to amplify up to 30kb):

 

KOD Hot Start

Qiagen LongRange

Elongase (did not work that well on very high GC templates)

TaqPlus

Pfu Ultra

LongAmp Taq

 

There were a couple more, but these were those that I used most.

BTW, 4 kb is often still within the range of most "normal" proof-reading polymerases. The yield might be a bit lower, but one could use them most of the time, too. I have successfully amplified ~4.5 kb (not high GC) with Pfx, Pfu and PWO (at least that are those that I remember).

Just to say thanks for the suggestions, we ended up going with Phire, Phusion, KAPA Hifi and a standard Taq. Turns out the Phusion from Finnzymes worked best, plus we've already got some more in the freezer.

Right, I forgot about Phusion. I think I had only used it a few times for relatively small fragments (~3kb) but with high (~75%) GC. Switched to KOD as it gave me more consistent results.

 

But having it in the freezer is usually the best reason to use it