I followed the standard SYBR Green Protocol for doing a qPCR. For which I used
- 10 uL of 1X SYBR Green Master Mix
- Forward Primer and Reverse Primer (each at a final conc. = 8.5 uM)
- Template (unknown concentration from an aptamer selection)
- Made to 20 uL with water.
Performed the PCR amplification using
- 95 C Initial denaturation
- 95, 68, 72 - cycled 35 times.
But, one set of sample from my aptamer selection (probably high GC content) did not amplify at all. I checked on an agarose gel too.
My primer conc. is intentionally high - I have always amplified with such concentrations and it works perfectly using Vent Polymerase.
My annealing temperature is also high at 68 C because my aptamer sequences tend to be GC Rich.
Using Vent polymerase my amplification is very strong
On reading notes from vendors, I found out that the polymerase in the mastermix to be some variant of Taq Polymerase (varies from vendor to vendor) and also it is known to poorly amplify GC Rich Sequences.
Hence, I wanted a suggestion from anyone working with aptamers, or if you have experience working with GC rich sequences on how I could amplify my sequences.
Much appreciated if I could have an answer before next week.
Thanks a lot for your help!!!