Hi there,

Have a go with the Phusion enzyme from ThermoFisher (Fermentas): it is cheap, rapid and Hi-Fi!

Thank you, I'll try!!

I agree with Dominique. Phusion has worked well in my hands, and in the cases where Phusion did not perform well I was lucky with Herculase II (Agilent).

Good luck

as long as I remember Phusion enzyme leaves blunt ends, so in order to use the pGEM-T vector you will have to add the A ovenhangs. You can find protocols for A tailing using regulat taq polimeraze adding only dATP instead of the four bases.

You can also try a mix of proofreading enzyme and regular Taq (usually 1:10).

Phusion works well, we have also had good luck with Bio-X-Act. There are now many hi fidelity enzymes available. You will have the A overhang issue with most unless you use one of the mixes. However you could also just add restriction sites to your primer and clone directly that way.

Presumably you know the sequence to expect. In that case, I prefer to have it synthesized (with warranty for correctness) and test it myself by sequencing.

At the bottom line, this save lots of time, cost and frustration.

Alternatively, try to find a way to obtain the correct/expected sequence by assembling pieces from individual clones that you already have sequenced (and have had a look at the raw data of the sequencing experiments, don't trust the text file you get from the base caller).

Hi, 

i am using the Taq platinum on regular basis to generate 2 kb fragments without any problem. can you describe the procedure that you use to amplify your sequence and what are the GC contents of your sequence ?