First Strand Synthesis with Reverse Transcriptase (M0253)
New England Biolabs
Published: 2022-02-16 DOI: 10.17504/protocols.io.bddyi27w
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Abstract
This protocol is for First Strand Synthesis with M-MuLV Reverse Transcriptase (M0253).
Before start
Prepare the following solutions:
10X RT buffer:
| A | B |
|---|---|
| Tris-HCl (pH 8.3 @ 25°C) | 500 mM |
| KCl | 750 mM |
| MgCl2 | 30 mM |
| DTT | 100 mM |
dNTP mix (2.5 mM each in water titrated by Tris-HCl to pH 7.0)
Steps
1.
In a sterile microfuge tube add the following:
| A | B |
|---|---|
| Reagent | Volume |
| RNA solution | 0.5-2 µg (total RNA) or 50-100 ng (PolyA-selected RNA) |
| Primer (Oligo-dT primer at 40 µM or Random nonamer N9 at 40 µM) | 2 µL |
| dNTP mix (2.5 mM each in water titrated by Tris-HCl to pH 7.0) | 4 µL |
| nuclease-free H2O | to final volume of 16 µL |
2.
Heat for 3-5 minutes at 65°C-80°C.
3.
Spin briefly and place promptly On ice.
4.
Add:
2µL
1µL
1µL
Final volume: 20µL
5.
Incubate at 42°C for 1h 0m 0s.
6.
Inactivate enzyme at 90°C for 0h 10m 0s.
7.
Store products at -20°C or proceed to next step(s).
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