First Strand cDNA Synthesis Kit using ProtoScript II Reverse Transcriptase (M0368)
New England Biolabs
Published: 2022-04-12 DOI: 10.17504/protocols.io.x14egnmmv5dy/v2
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Abstract
This protocol explains methods for the "First Strand cDNA Synthesis Kit using ProtoScript II Reverse Transcriptase (M0368)".
Before start
Steps
1.
In a sterile microfuge tube add:
| A | B |
|---|---|
| RNA solution 1ng-1 µg total RNA or 1-100 ng polyA-selected RNA Primer (50 µM dT or 60 µM Random Primer Mix) | 2 µl |
| 10 mM dNTP mix | 1 µl |
| Nuclease-free H2O | to final volume of 12 µl |
2.
Optional: Heat for 3-5 minutes at 65°C-70°C. Spin briefly and place promptly 65On ice.
3.
Add the following (bringing to 20 µl final volume ):
| A | B |
|---|---|
| 5X ProtoScript II RT Reaction buffer | 4 µl |
| 0.1 M DTT | 2 µl |
| Murine RNase inhibitor (40 U/µl) | 1 µl |
| ProtoScript II Reverse Transcriptase (200 U/µl) | 1 µl |
| Final volume | 20 µl |
4.
If Random Primer Mix is used, an incubation step at 25°C for 0h 5m 0s is recommended before the 42°C incubation in the next step.
5.
Incubate at 42°C for 30 minutes-1 hour.
6.
Inactivate enzyme at 80°C for 0h 5m 0s.
7.
Store products at -20°C or proceed to next step(s).
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