Difference between revisions of "First Strand cDNA Synthesis (AB Kit)"
From Bridges Lab Protocols
(adjusted quantity of RNA added) |
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* Add 10 uL Master mix per well into tube or plate | * Add 10 uL Master mix per well into tube or plate | ||
| − | * Add | + | * Add 10 ug RNA in a final volume of 20 uL to each well. Since results will be normalized in the end, standardizing RNA quantities at this step is unnecessary. Total volume can be adjusted down if RNA is in short supply. |
* Run the PCR Program '''AB Reaction Protocol'''. Takes ~2h: | * Run the PCR Program '''AB Reaction Protocol'''. Takes ~2h: | ||
** 10 min at 25C | ** 10 min at 25C | ||
** 120 min at 37C | ** 120 min at 37C | ||
** 5 min at 85C | ** 5 min at 85C | ||
Latest revision as of 15:16, 24 July 2019
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 10 ug RNA in a final volume of 20 uL to each well. Since results will be normalized in the end, standardizing RNA quantities at this step is unnecessary. Total volume can be adjusted down if RNA is in short supply.
- Run the PCR Program AB Reaction Protocol. Takes ~2h:
- 10 min at 25C
- 120 min at 37C
- 5 min at 85C
