Jun 26, 2019
Version 3
Reverse transcription using SuperScript IV V.3
- 1Harvard Medical School
Protocol Citation: Amin Mahpour 2019. Reverse transcription using SuperScript IV. protocols.io https://dx.doi.org/10.17504/protocols.io.4sugwew
Manuscript citation:
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: June 26, 2019
Last Modified: June 26, 2019
Protocol Integer ID: 25140
Keywords: Using SuperScript IV to produce cDNA from RNA
Guidelines
Use high quality RNA as the substrate.
Keep RNA on ice at all times.
Use RNase free water.
Materials
MATERIALS
SuperScript™ IV Reverse TranscriptaseThermo Fisher ScientificCatalog #18090050
Mix the following reagents from the kit. Please scale up if more RT reaction is desired.
| Reagent | Amount (uL) | |
| Primer (Random or dT) | 0.5 | |
| dNTP (10mM) | 1 | |
| RNA | 11 |
Incubate the mixture at 72 °C for 00:02:00 . Then, incubate samples on ice for few minutes.
Note
This step allows denaturation of RNA and proper priming for the downstream cDNA synthesis.
Mix the following reagents from the kit. Please scale up if more RT reaction is desired.
| Reagent | Amount (uL) | |
| RT Buffer (5x) | 4 | |
| DTT(10mM) | 1 | |
| RNase Inhibitor | 1 |
Add the 6uL to the 12.5uL mix from Step 3.
Incubate the samples at 37 °C for 00:05:00 . Then, add 1.5uL SuperScript RT IV enzyme to the reaction and mix well.
Incubate the samples using the following incubation settings:
| Temp (C) | Time (minutes) | |
| 25 | 5 | |
| 45 | 40 | |
| 55 | 10 | |
| 75 | 10 |
Add 1uL RNase H to the cDNA samples and incubate at 37 °C for 00:20:00 .
Dilute the cDNA samples using Nuclease free water.