Hello,
I am genotyping by PCR and am having difficulty amplifying a 2kb fragment. Increasing the Taq concentration improves the signal, but then there are a lot of non-specific bands. Any advice for amplifying 2kb+ amplicons from worm lysate? Should I try ethanol precipitation? I am thinking about using phusion, but it is an expensive alternative for screening.
Best,
Dan
You definitely shouldn’t have to resort to Phusion for just plain old genotyping. I think you should try a different set of primers or play around with the annealing temperatures to optimize it.
Try adding different concentrations of DMSO (1-2%) also you can change amount of MgCl2 in buffer. You can change the annealing temperature. Or order new primers as Snug suggested. There are many ways to get of non-specific primers by changing PCR conditions slightly. Good luck!
First, just bad luck with ghost bands, depending on your primers. Second, I always shoot for smaller products: 200-600 bp is my preferred range, and that is more likely to give reproducibly robust product.
If you need to get rid of ghost bands, try raising your Tm, or optimize by doing a Tm gradient. But good primer design and smaller products will generally do the trick.