Oct 16, 2019
First strand cDNA synthesis (ThermoScientific RevertAid)
- 1Wageningen University
- iGEM Wageningen 2019
Protocol Citation: Ben Kuipers 2019. First strand cDNA synthesis (ThermoScientific RevertAid). protocols.io https://dx.doi.org/10.17504/protocols.io.8bmhsk6
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: October 16, 2019
Last Modified: October 16, 2019
Protocol Integer ID: 28749
Abstract
The following protocol is optimized to generate first-strand cDNA for use in (q)PCR
Materials
MATERIALS
5X RT BufferThermo Fisher ScientificCatalog ##B91
dNTP Mix 10 mM eachThermo Fisher ScientificCatalog ##R0191
Water, nuclease free
RiboLock RNase Inhibitor Thermo Fisher ScientificCatalog ##EO0381
RevertAid Reverse Transcriptase (200 U/µL)Thermo FisherCatalog #EP0442
Oligo(dT)18 PrimerThermo FisherCatalog #SO131
Before start
Mix and briefly centrifuge all reagents after thawing, keep on ice.
Add reaction components into sterile, nuclease-free tube on ice in the indicated order:
| Template RNA | 100 ng ( 1pg - 5 µg) | |
| Oligo(dT)18 | 1 µl (100 pmol) | |
| Water, nuclease-free | to 12 µl |
Optional: If the RNA template is GC-rich or is known to contain secondary structures, mix gently, centrifuge briefly and incubate at 65 °C for 5min. Chill on ice, briefly centrifuge again and place on ice
ice.
| 5X RT Buffer | 4 µl | |
| RiboLock RNase Inhibitor | 1 µl (20 U) | |
| RevertAid RT (200 U/µL) | 2 µl (400 U) | |
| 10 mM dNTP Mix | 1 µl | |
| Total volume | 20 µl |
Mix gently and centrifuge briefly.
| 5 min | 25 °C | |
| 60 min | 42 °C (For GC-rich RNA, the reaction temperature can be increased to 45 °C) | |
| 5 min | 70 °C |
The cDNA product is now ready for downstream applications