high fidelity PCR polymerase - ask for help! (Sep/11/2008 )
I'm doing amplifications of 300 bp but almost no PCR band can be observed. I am using Phusion master mix from NEB, and AT content of my template is around 80% ... Is there any other high fidelity DNA polymerase is good for high AT DNA template?
Thank you in advanced.
Low GC templates can be tough. Stick with Phusion - it's the best imo. You can add more of it but there is usually enough if you are adding 0.5 uL per 50 uL reaction. Add an additional 2.5 mM MgCl2 to your reaction to encourage stronger primer annealing. Also add more template to increase the likehood the primers will anneal to their target. If you get any hint of a PCR product then isolate it and re-amplify it. If these things don't work, design primers with longer annealing sites and a larger Tm (i do around 70C for all my primers). One of the issues in AT-rich PCR is getting the primers to anneal or stay annealed to the template. A GC-clamp at the 3` end of the primers may help. If you do order new primers, try all the primers (new and old) in the same reaction or different combinations.
You should also try to do a gradient PCR to see if other temps. might be better
Lower your extension temperature to 64-66. See:
Su XZ, Wu Y, Sifri CD, Wellems TE.
Reduced extension temperatures required for PCR amplification of extremely A+T-rich DNA.
Nucleic Acids Res. 1996 Apr 15;24(8):1574-5.