Problems with Invitrogen?

[steveF]

Hello.

Fot the past few weeks I have been trying to amplify several fragments using Invitrogens platinum pfx. I got very little success, either no amplification at all or very faint bands. This was using template/primer pairs that I had shown to work with taq. After changing everything, I finally did a definitive exp comparing the pfx against pfu-ultra and herculase. both of the latter gave good strong single bands. pfx gave nothing at all.

This is not the first time i have had problems with invitrogen products. I simply do not believe the claims that they make for their products or their claims of qualit control.

Avoid pfx platinum. It is a very poor product.

Steve

[SebaQ]

The problem you address it's a common one we have at our lab, too... this enzyme amplifies whenever it wants to, and most of the time doesn't amplify at all.

The thing is, I do need to clone some genes we have in mind, and it has to be with proofreading activity... what enzyme should I use?

[steveF]

KOD hot-start. Good proof reader with a very good processivity rate. And not too expensive either.

[mmorgan]

I use Platinum Pfx almost exclusively for cloning and it usually performs well for me (However I do always run 4 replicate reactions at 55C and 60C annealing and +/- enhancer solution and often amplification is very temperature and enhancer dependent, even when using plasmid templates).

I certainly have had instances where Taq could amplify products that Pfx could not (This was in a 5' RACE experiment). I suppose the times that Pfx has failed for me have not been frequent enough for me to switch to a different enzyme, but I would certainly be interested to know about better alternatives (I have tried Pfu-Ultra and I also found it to work very well).

One Pfx protocol modification that I use is that I always use the reaction buffer at 2x instead of 1x.

Also, and I have found this to be a VERY critical point, For amplification of plasmid templates, cut the extension time way down. I use 2 seconds at 68C for anything less than 500bp. For larger amplicons I use about 30 sec / 1kb. I think that the polymerase extends very rapidly, so it helps to reduce extension times (I have had some instances where this made the difference between having a smear without a distinct product and having a strong single band).

Does anyone know why Pfx has the problem that it seems to fail now and again? I was under the impression that Pfx is a modified version of KOD. Perhaps some of the amino acid substitutions are deleterious under certain conditions?