Sep 13, 2022
cDNA synthesis
- 1Vall d'Hebron Research Institute
Protocol Citation: Miquel Vila 2022. cDNA synthesis . protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg3948qg25/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: September 13, 2022
Last Modified: September 13, 2022
Protocol Integer ID: 69944
Abstract
High-Capacity cDNA Reverse Transcription Kits(#4368814, Applied Biosystems - ThermoFisher)
cDNA synthesis
cDNA synthesis
Prepare the 2X master mix (ul per sample):
- 2ul RT Buffer
- 0.8ul 25X dNTP Mix(100mM)
- 2ul Random primers
- 1ul Multiscribe Reverse Transcriptase
- 4.2ul Nuclease free water
Take 0.5ug of RNA in a maximum of 10ul of sample volume
Mix the 2X master mix (10ul) with the RNA samples (10ul) and mix by pippeting
Centrifuge the plates to spin down all the reagents.
Keep the samples on ice
Program the thermal cycler to 25C for 10min 37C for 120 mintues 85C for 5 minutes and 4C until finished.
Store the cDNA samples at -20C for short-term storage or -80C for long-term storage (years).