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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 ℃ for 30 minutes.
6. Follow by PCR reaction cycles.
7. Analyze the samples on a 1-2% agarose gel.
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Detecting DNA 