SOP for cDNA synthesis by RT (Reverse Transcription) Promega kit
Malu G Tansey
Published: 2024-02-27 DOI: 10.17504/protocols.io.8epv5xqkjg1b/v1
Abstract
SOP for cDNA synthesis by RT (Reverse Transcription) Promega kit
Steps
Before Begining:
1.
Take from the kit: OligodT, RT buffer (5x), MgCl2, dNTP mix, and H2O to thaw- you can keep them on ice or at RT for a little while.
Protocol:
2.
Prepare per sample:
| A | B |
|---|---|
| total RNA (1 pg-1ug) | max vol 4ul |
| Water | Fill upti 4ul |
| OligodT | 1ul |
| Final Volume | 5ul |
3.
Heat samples at 70°C for 5 min (prepare mix for step 4).
4.
Snap freeze samples at +4°C or on ice for a minimum of 5 min.
5.
Prepare RT mix (vol per samples, calculate for all your samples + 1):
| A | B |
|---|---|
| RT buffer (5X) | 4ul |
| MgCl2 (25 mM) | 3ul |
| dNTP Mix (10 mM) | 1 ul |
| Rnasine* | 0.5 ul |
| Rtase* | 1 ul |
| Water | 5.5ul |
| final volume | 15 ul |
- add them in the last step before adding the mix to each sample. Meanwhile keep them at 20°C.
6.
Brief centrifugation.
7.
RT program
| A | B |
|---|---|
| hybridization | 5 min at 25°C |
| elongation | 60 min at 42°C |
| inactivation of the enzyme | 15 min at 70°C |
8.
Add H2O to bring final concentration to a desired amount – this will likely depend on the abundance of your targets.
9.
Once it is done, cDNA can be stored at -20°C
