I have a serious trouble here: I am trying to qPCR a ~300bp region with 72% GC content without any luck so far. I was running a load of optimizing reactions: including temperature gradient for anealing with addition of DMSO 0; 2 and 5% . I am using the Brilliant II SYBRŪ Green QPCR Master Mix from Stratagene with Hot Start Polymerase (therefore initial denaturation step was 10min at 95C), Mix has 2.5mM MgCl2, which is a relatively high concentration. The only thing I haven't tried yet is the TD-PCR, but it doesn't work for quantitative purpose...
Any ideas, suggestions to continue with? Perhaps raising the denaturation temperature/time? The melting temperature of region is higher than 100C, but won't it kill polymerase even though it is a hot start enzyme?
Could it also be a primer issue: one of the primers has a Tm around 62, the other one around 72 (and 73% GC content)?
Any other thoughts???
The only thing I know for sure: it shouldn't be a template issue, another target works perfectly.
Thanks in advance,