We are trying to do qPCR with the following template and primer set,
GG CCT CCT CCA ATT AAA GAA ACG CAA CTT TAG AAA AGA AAA CGA TCT AAG CTC TAC GAT GTA AGA GTA CCC AAA TAA CGG TTC AC is the templateTm = 70
FW primer: GGC CTC CTC CAA TTA AAG AA Tm = 51
RV primer:GTG AAC CGT TAT TTG GGT AC Tm = 51
we are getting an amplification plot with CT values around 4-11 for samples 0.5 nmol and 0.05 nmol concentration , but we also see the amplification in NTC and a Ct value at 12, we tried many times with several condition but results are same !! melt curve shows only one peak 77, also gel show a band less than 100 (matching with the expected product).
So how we can eliminate the NTC Ct value ?