Feb 07, 2022
Reverse Transcription of Phage RNA
This protocol is a draft, published without a DOI.
- 1UCPH
- FOOD Micro UCPH
Protocol Citation: Frej Larsen 2022. Reverse Transcription of Phage RNA. protocols.io https://protocols.io/view/reverse-transcription-of-phage-rna-b4p4qvqw
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: February 04, 2022
Last Modified: February 07, 2022
Protocol Integer ID: 57820
Abstract
Reverse transcription (RT) is the process of turning ssDNA into cDNA using the reverse transcriptase enzyme. This protocol uses the RT reaction to turn ssRNA and dsRNA into ssDNA and is specifically used for virome analysis.
Materials
SuperScript IV VILO Master Mix from Invitrogen. Can be bought here: https://www.thermofisher.com/order/catalog/product/11756500?SID=srch-srp-11756500
Add 16 µL sample to a PCR tube and denature RNA by heating to 95 °C for 00:05:00 on a ThermoCycler
5m
Centrifuge for 00:00:10 and put on ice
10s
Add 4 µL of the 5X master mix to the PCR tube, then run the following program on a ThermoCycler:
- 25 °C for 00:10:00 (primer annealing)
- 50 °C for 00:10:00 (reverse transcription)
- 80 °C for 00:05:00 (enzyme inactivation)
25m
Centrifuge for 00:00:10
10s
Purify by bead cleaning and elute in 10 µL MQ water