Standard vs High fidelity polymerase, please help - No results with high fidelity (Jul/06/2006 )

Hi, I ask for help desperately. I'm trying to join a tag (15bp) to my protein sequence. My purpose is joining this tag and then use the sequence+taq to link to another protein.

I've done successfully with a standard polymerase (Promega) at the first try but when I try a high fidelity polymerase (with its appropriate buffer and protocol) I don't get reproducible results. I've tried everything: different Mg, template, primers, number of cycles, more units of enzyme, DMSO and I only got a faint band when I use a specific Mg and template concentration and 40 cycles.

When I used higher Mg concentration I got nothing, should I have obtained the same band and maybe anothers because of unspecificity?

When I used more enzyme, I got a single higher band. (????)

My template is a plasmid and contains EDTA because I used TE buffer. However, I used another with no EDTA and I got nothing again.

I use a plasmid with my protein sequence because I want to obtain exactly the same sequence to express it later and this is the reason because I want to use a high fidelity enzyme.

Now I'm thinking of giving up and just doing with the standard polymerase. Then trust that when I do ligation+transformation I got a colony not with the right DNA sequence but with the right aminoacid sequence (I mean with some silent mutations) which is the only important part when you want to express in a cell line, isn't it?

I'm looking forward to hearing your suggestions and advice.

PD: The enzyme worked well with other sequence

ok well if you have a amplification with the 'standard' polymerase, go for it. (but what is the length of the sequence?)
You're right, having silent mutation is ok regarding protein aminoacids. But in cells, codon usage is different for each combination. I mean some triplets that encode for leucine are more used as others. And so the corresponding tRNA is supposed to be more abundant in the cell.
but that shouldn't matter you at first.

Thanks for your prompt reply. I needed a vote of confidence from one of the experts. So, I go for it. The protein sequence is not long, just 700bp so the likelihood of misincorporation will not be big (at least, it is what I hope).

However, could you (or anyone) give me some advice about the potential causes of my problems? I would like to explain to someone else why this happened and to be honest, I don't have a clue (to say rubbish high fidelity enzyme is a little bit rude, isn't it)

things i think are about buffers. There is surely a difference in you buffers that may explain the changes. high fidelity polymerases donesn't like too much the tails in primers.

Change to other brand of high fidelity polymerase.
I have found some brands of high fidelity polymerase's is not stable, I have use one brand that can amplify >3kb fragment at begining(even better than normal Taq), while failed to amplify even for 1 kb fragment using the same brand polymerasedifferent ordered later...

by the way 700bp is not too long for normal Taq, you might sequence 2-3 clones and will found at least one is correct.

In fact I always check sequence for such PCR clones even using high fidelity polymerase.

I've noticed too that the high fidelity polymerases work poorly when the primers contain tag or restriction enzyme site sequences that are not complementary to the target. One enzyme that is particularly robust is KOD Hot-Start polymerase...it usually works well when other high fidelity enzymes fail.

Since your amplified product is so small, I would do like rye and just sequence a few to see if you got lucky and didn't get mutations. However, if you did 40 cycles, that leaves a lot of time for mutation. I would use do a PCR with a high mass of template (10 to 100 ng of plasmid) and a low number (12 to 15) of cycles to increase your chances of getting your product without mutation.

Even easier would be to use Stratagene's QuikChange kit or protocol to insert the 15 bp. It uses Pfu Turbo, which is a high fidelity enzyme. To make the mutagenesis more efficient, use the Two-step modification described in BioTechniques 26:680-682 (1999) for reliable site-directed mutagenesis and insertion or deletion of sequences. You would use one primer pair, PCR introduce the 15 bp, DpnI digest, transform competent cells, and then screen colonies. If you can knock in or abrogate a restriction site using your mutagenesis primers, you can initially screen clones by restriction analysis. The procedure is usually over 50% successful, so just screening by sequencing isn't out of the question.

Good luck.