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I have worked with human GC rich regions only, but 5% DMSO was the best fit for most, you can try up to 1 0%. It still amplified but didn't had such bright product. In my case 1 % was not enough, if you have more GC rich template, maybe 3 % is still not enough, try more concentrated.
What about the Primer3 Tm values? They usually work for me with Ta -4 degrees from this calculation. Tm's can vary a lot, that's why I'm asking about algorithm I'm comfortable with.
And another thing, did you checked the DNA is free of inhibitors? Do you have any working assay on the same species, so you can exclude potential problems with template?
#140669 PCR no amplification
Posted
on 04 September 2012 - 12:36 PM
