Feb 27, 2024
SOP for cDNA synthesis by RT (Reverse Transcription) Promega kit
- 1College of Medicine |University of Florida
Protocol Citation: Malu G Tansey 2024. SOP for cDNA synthesis by RT (Reverse Transcription) Promega kit. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5xqkjg1b/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 28, 2024
Last Modified: May 31, 2024
Protocol Integer ID: 94277
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson’s (ASAP)
Grant ID: ASAP-020527
Abstract
SOP for cDNA synthesis by RT (Reverse Transcription) Promega kit
Before Begining:
Before Begining:
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:
Protocol:
Prepare per sample:
| A | B | |
| total RNA (1 pg-1ug) | max vol 4ul | |
| Water | Fill upti 4ul | |
| OligodT | 1ul | |
| Final Volume | 5ul |
Heat samples at 70°C for 5 min (prepare mix for step 4).
Snap freeze samples at +4°C or on ice for a minimum of 5 min.
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.
Brief centrifugation.
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 |
Add H2O to bring final concentration to a desired amount – this will likely depend on the abundance of your targets.
Once it is done, cDNA can be stored at -20°C