RT-PCR --- one-step
This protocol uses C. therm. Polymerase One-Step RT-PCR system from Roche.1. Set
This protocol uses C. therm. Polymerase One-Step RT-PCR system from Roche.
1. Setup master mix 1:
| 25 mM dNTP | 0.8 m l |
| DMSO | 2.5 m l |
| 100 mM DTT | 2.5 m l |
| RNase Inhibitor (40 U/m l) | 0.5 m l |
| forward primer (10-20 m M) | 1 m l |
| reverse primer (10-20 m M) | 1 m l |
| template RNA (50 pg-1 m g) | 1 m l |
| nucelase-free ddH2 O | 15.7 m l |
2. Setup master mix 2:
| 5X RT-PCR buffer | 10 m l |
| C. therm. polymerase mixture | 2 m l |
| nucelase-free ddH2 O | 13 m l |
3. Mix 25 m l master mix 1 and 25 m l 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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