I am trying to standardize a taqman assay using a probe of 24mer with Fam as reporter and Tamra as the quencher, the probe is from SIGMA and is at stock concentration of 100UM made with TE buffer. The Calculated Tm of the probe is 65.3C and those of the primers are 63C. Am using the ABI Universal Taqman reagent with UNG for the assay. The conditions are 50C for 2 mins, 95C for 10mins and 35 cycles of 95C for 15sec and 60C for 1min.
I use diluted plasmids from copy number starting from 10 power 7 to 10 power 2 in a ABI 7500 machine. I have previously checked all the diluted plasmids and primers with SYBR green and they have given me r2 of 0.999 and efficiency of 98.9%. But when I use the same set of primers and diluted plasmids for the Taqman assay it does not work. There is no amplification see at all. When I run the products on a gel I see clear bands without any primer dimer or non specific bands.
I am using :
8]Taqman MasterMix 2x 12.5ulprimer (20pmoles/ul) 1.0ul
primer (20pmoles/ul) 1.0ul
probe (20uM) 0.2ul
RNase-free H2O 8.3 ul
I have played with increasing and decreasing the probe concentration, anneling temperature went down to as low as 50C and as high as 62C with 2 degree increments every tube had good bands as observed on a gel but no amplification was seen on the plot.
Is it a problem with the probe?