Hi Judit,

I worked once with Phusion but had no problem. I think if your primers and the quality of your DNA are OK there should be no issue. Indeed, 45% is not high GC but you should look at GC content on small stretches. I suggest you add betaine up to 1.5 M- 2.5 M and try again. Alternatively, you could also use Platinum Pfx from Invitrogen. It has both proofreading activity and strand displacement capacity.

I have used Phusion High-fidelity Polymerase with HF buffer and addition of 5 % -10 % DMSO to avoid problems of high GC content template and secondary structures.

I have used long distance PCR program: 98 0C – 30 s (98 0C – 10 s; 65 0--60 s; 72 0 – 200 s) x25; 72 0C – 10 min; 4 0C –hold. and 50 uL reaction volume.

Mainly, I followed manufacturer's protocol and I've got even 6000 bp fragment as expected, without any problems.

I use Phusion (and other enzymes such as PrimeStar) almost always with GC buffer. It works much better compared to the HF versions. Since there is not enough GC buffer included with most enzymes, I generally buy a bit extra of it. PrimeStar is the exception that you also buy as the GC version. Both Phusion (and now PhusionII) and PrimeStar are superior enzymes (compared to many others). I did extensive comparison between 7 or so enzymes (including Pfu, PfuUltra, Herculase, iProof, HotStar HiFidelity)

I typically use the GC buffer only for genes with >60% GC content. I often use Phusion with HF buffer for total vector amplification (up to 10Kb) with no issue. Just check that your extension time is long enough. For long products I often use the high end of the range they give you. If your sure it's a PCR problem, try different extension times and longer initial denaturation time.

Thank you for the answers! I got really some good new ideas to try, so we will see.

If your GC is content is nor very high, its better to use HF buffer and try. If its not working use GC buffer and try DMSO conentrations between 3-10%.I have been using Phusion polymerase and it works almost always..