PCR polymerases

[MedGen]

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.

[CharonY]

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).

[MedGen]

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.

[CharonY]

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