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!

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