We have a 2X mastermix of taq DNA polymerase, SYBR Green, and dNTPs. Primers are stored at 100μM and used at final concentrations between 200-600nM. The PCR mix will be as follows:

qRT-PCR using Quanta PCR mastermix

Component Volume
Quanta PCR master mix 5 μL
Primers (Fwd&Rev) at 600nM each 3 μL
cDNA 2 μL
Final Volume 10 μL

The following cylcing conditions are used.

2 min at 50°C
10 min at 95°C
40 cycles of:
15 sec at 95°C
1 min at 60°C
Melting curve analysis
15 sec at 95°C