First Strand cDNA Synthesis (AB Kit)
From Bridges Lab Protocols
Materials
- Purified RNA
- High Capacity cDNA Reverse Transcription Kit without RNAse inhibitor (AB 4368813 for 1000 reactions, see http://products.invitrogen.com/ivgn/product/4368813)
Protocol
- Generate Master Mix Containing:
| Component | Per Reaction | Per 10 Reactions |
|---|---|---|
| 10X RT Buffer | 2 | 20 |
| 25X dNTP Mix (100 mM) | 0.8 | 8 |
| 10X RT Random Primers | 2 | 20 |
| MultiScribe Reverse Transcriptase | 1 | 10 |
| Nuclease Free Water | 4.2 | 42 |
- Add 10 uL Master mix per well into tube or plate
- Add 0.1-10 ug RNA in a final volume of 10 uL to each well
- Run the PCR Program AB Reaction Protocol. Takes ~2h:
- 10 min at 25C
- 120 min at 37C
- 5 min at 85C
