Setting up quantitative real-time PCR qRT-PCR
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cDNA synthesis from RNA
✓ Reaction mix per sample
| ☐ | 5X RT buffer | 4 µL |
| ☐ | dNTP-Mix | 2 µL |
| ☐ | reverse transcriptase | 1 µL |
| ☐ | random hexamer primer (160 µM), primer #312 | 1.25 µL |
| ☐ | oligo dT18 (100 µM); primer #945 | 0.1 µL |
| ☐ | RNA | 1-2 µg |
| ☐ | RNA Inhibitor | 0.5 µL |
| ☐ | – | – |
| ☐ | RNAse-free dd H2O | up to 20 µL |
- Incubation of sample for 1 h at 37°C
- Heat-inactivation of enzyme at 70°C for 15 min
qRT-PCR
✓ Reaction mix per sample
| ☐ | H2O | 18.3 µL |
| ☐ | 10x Taq/Pfu buffer | 2.5 µL |
| ☐ | Primer 1 | 0.5 μL (10 pmol) |
| ☐ | Primer 2 | 0.5 μL (10 pmol) |
| ☐ | Template | 2 μL (~10 ng) |
| ☐ | dNTPs | 0.2 µL |
| ☐ | SYBR Green | 0.5 µL |
| ☐ | Taq Polymerase | 0.5 µL |
| ☐ | Total | 25 µL |
Important notes:
- optimized for AppliChem SYBR Green CAS# A8511
- SYBR Green in higher concentration inhibits polymerase activity