10.2.8.5. Real-time qPCR

The SuperScript-III, Thermoscript and rTth cDNA reactions all enter a separate (TwoStep) PCR protocol, which can be conveniently adapted to real-time qPCR, using a real-time qPCR kit containing SYBR-green:

1. Mix:
1.1.  3 μl      cDNA (column purified, or diluted 1/10),
1.2.  0.4 µl   10 μM tag primer               (0.2 μM final concentration),
1.3.  0.4 µl   10 μM virus-specific primer (0.2 μM final concentration),
1.4.  0.4 µl*  10 mM dNTP*                   (0.2 mM final concentration*),
1.5.  x µl      Buffer + SYBR-green         (as per manufacturer),
1.6.  y µl      nuclease-free water,
1.7.  z µl      Taq polymerase                 (as per manufacturer),
1.8.  20 µl    total volume.
(* dNTPs are often included in the optimized buffer)
2. Incubate in real-time thermocycler:  
         2.1.  5 min:95°C,
         2.2. 35 cycles [10 sec:95°C - 30 sec:58°C - read].     
3. For Melting Curve analysis of the products, incubate:
         3.1.  1 min:95°C,
         3.2.  1 min:55°C,
         3.3.  5 sec:0.5°C:read from 55oC to 95oC.