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RT-PCR --- one-step

This protocol uses C. therm. Polymerase One-Step RT-PCR system from Roche.

1. Setup master mix 1:

25 mM dNTP 0.8 ml
DMSO 2.5 ml
100 mM DTT 2.5 ml
RNase Inhibitor (40 U/ml) 0.5 ml
forward primer (10-20 mM) 1 ml
reverse primer (10-20 mM) 1 ml
template RNA (50 pg-1 mg) 1 ml
nucelase-free ddH2O 15.7 ml

2. Setup master mix 2:

5X RT-PCR buffer 10 ml
C. therm. polymerase mixture 2 ml
nucelase-free ddH2O 13 ml

3. Mix 25 ml master mix 1 and 25 ml master mix 2.

4. Overlay with mineral oil if required.

5. Program the thermocycler such that the first cycle is for RT reaction at 60-72 C for 30 minutes.

6. Follow by PCR reaction cycles.

7. Analyze the samples on a 1-2% agarose gel.