Long Primers for PCR - (Mar/12/2010 )
Hello everyone I was hoping to get some tips from your experience.
I am using primers that are longer than what i tend to design. (30-40bp) for PCR. My sequence is very AT rich and I am having to make long primers to get higher tm's (55-60C). I tried shorter primers with tm's in the 50-55 and the PCR did not work.
Can you guys share some of experiences with longer primers in PCR.
I have products ranging from 400bp to 2000bp
The hairpins, self-primer, hetro-primer are good (more positive than -9)
I do have a lot of repeat sequences in the primers, 3 or 4 single base repeats mostly A's and T's
I appreciate any tips/experieces. Thank you.
It sounds as if you are working in a low GC organism. Be aware that PCRs can fail because of 25-35 bp regions of extremely low GC content in the template, where the extending strand falls off of the template strand at normal (72) extension temperatures. Use lower temperatures for extension, down to 64, and lengthen the extension period a little to get around this problem.
I am also working on an organism with low GC content. my primers also tend to be longer than usual ~30 bp....
although extension temperature of 68-72 works fine in my case, I have to lower my annealing temp. considerably as compared to the Tm of my primers.....sometimes as low as 45 degrees also. Phusion polymerase works really well in my case....Hope this helps....