help me to choose a high fidelity taq polymerase - (Jul/20/2005 )

Hi, all,
Do you have some suggestions on taq polymerase? i have been using the Expand High Fidelity taq. But it seems the enzyme gives error quite frequently. So far, I have pcr out 10 regions. Five of them have error in sequence. (my pcr products are all smaller than 1Kb). I have to redo it. This takes me too much time. Is there a better enzyme? Please help me out. Thanks!!

Quiagen Proofstart DNA polymerase. It has been giving me very reliable reads for 1.5Kb+.

Do not neglect the 95C 5 min denaturation step.

Also keep in mind that the conditions that you do the PCR under are probably more important than the enzyme used. Key things that influence fidelity are:

1. Mg conc (low is better)
2. dNTP conc (low is better)
3. dNTP balance (balanced is better)
4. Extension time (less is better)
5. Cycle number (less is better)

You can try for instance:

1. Roche's Expand hign fidelity PLUS (has 2 fold lower error rate than EHF)
2. Invitrogen's platinum taq high fidelity (2 fold lower error rate than EHF)
3. Invitrogen's platinum pfx (has about 8 fold lower error rate than EHF)
4. Stratagene's Pfu-Ultra (has about 6 fold lower error rate than EHF)
5. Finnzyme's Phusion (has about a 15 fold lower error rate than EHF)


These are the one's I know of, but probably every company has a high fidelity polymerase.
But please read this thread as it deals about how when manufacturer's compare their enzymes the results aren't the same! So, I just gave you the error rates the company's are giving you, I can not guarantee you if they are correct (as far as I know there are no independent tests done on this matter).

And indeed, check all the mentioned PCR parameters!

I use pfu turbo for cloning, you can try that

What do you mean by dNTP balance?

If the nucleotides are not of equal molar ratio then this can increase the mutation rate. In fact this one way of doing PCR mutagenesis (ie use unbalance nucleotide concentrations).