4.7.3. Two-Step RT-qPCR

The following is a robust, standard qPCR protocol for amplifying and quantifying targets <400bp in length, using SYBR-green detection chemistry, and starting with a cDNA template:

  1. Mix:  
    3 μl    cDNA (pre-diluted 1/10, in nuclease-free water),
    0.4 µl           10 μM Forward primer (0.2 μM final concentration),
    0.4 µl           10 μM Forward primer (0.2 μM final concentration),
    0.4 µl*          10 mM dNTP*        (0.2 mM final concentration*),
    x µl    Buffer + SYBR-green (as per manufacturer),
    y µl    nuclease-free water,
    z µl    Taq polymerase (as per manufacturer),
    20 µl  total volume.
    (* dNTPs are often included in the optimized buffer)
  2. Incubate in real-time thermocycler:
          95°C for 5 min,
          35 cycles of:
    95°C for 10 sec,
    58°C for 30 sec* read (qPCR),
  3. For Melting Curve analysis of the products, incubate:
    95°C for 1 min,
    55°C for 1 min,
    +0.5°C increments for 5 sec, with reads from 55oC to 95oC.

 

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