Hello everyone!
My problem is that unfortunately I am getting a good Ct value in NTC (NFW). I have tried three different primer sets (Target of three different genes) with NFW from different sources and surprisingly each primer set returned with a good Ct values.
Believing that SYBR-green (fast) could have been contaminated so, I used a totally new SYBR-green reagent yet there was no significant change observed upon RT-PCR.
I ordered new set of primers for same genes thinking that might be stock primers were contaminated and repeated RT-PCR with new SYBR green and fresh NFW yet I see there is very mild yet not a big difference in Ct values.
I use gloves and filter tips all the time.
I will be very grateful if anyone can give me some useful suggestion or insight to fix this never ending problem.
Reaction volume per well= 20ul
Primer conc.- 1uM each (FP/RP)
amplicon size ≈ 70-80 bp
Gene 1 Ct= 19
Gene 2 Ct ≈ 26-28
Thanks!