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.
Important Notes:
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[1][3] (varies from vendor to vendor) and also it is known to poorly amplify GC Rich Sequences[2].
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!!!
