First Strand Synthesis Protocol with Reverse Transcriptase

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Overview

Materials needed:
10X RT buffer:
500 mM Tris-HCl (pH 8.3 @ 25°C)
750 mM KCl
30 mM MgCl₂
100 mM DTT

dNTP mix (2.5 mM each in water titrated by Tris-HCl to pH 7.0)

Oligo-dT primer (40 µM)

Random nonamers (40 µM)

RNase Inhibitor (10 U/µL)

M-MuLV Reverse Transcriptase (200 units/µL)

Protocol

1. In a sterile microfuge tube add:
   RNA solution  0.5-2 µg ( total RNA or 50-100 ng polyA-selected RNA)
   Primer (dT or N9)         2 µL
   dNTP mix                     4 µL
   nuclease-free H₂O to final volume of 16 µL
2. Heat for 3-5 minutes at 65-80°C. Spin briefly and place promptly on ice.
3. Add:
   10X RT buffer 2 µL
   RNAse inhibitor 1 µL
   M-MuLV Reverse Transcriptase 1 µL
   ______
   final volume 20 µL
4. Incubate at 42°C for one hour.
5. Inactivate enzyme at 90°C for 10 minutes.
6. Store products at -20°C or proceed to next step(s).