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Protocol: First Strand Synthesis Protocol with Reverse Transcriptase, NEB
New England Biolabs
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First Strand Synthesis Protocol with Reverse Transcriptase


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 然)

Random nonamers (40 然)

RNase Inhibitor (10 U/無)

M-MuLV Reverse Transcriptase (200 units/無)




Introduction





Protocol


  1. In a sterile microfuge tube add:
    RNA solution 0.5-2 痢 ( total RNA or 50-100 ng polyA-selected RNA)
    Primer (dT or N9)2 無
    dNTP mix4 無
    nuclease-free H2O to final volume of 16 無
  2. Heat for 3-5 minutes at 65-80°C. Spin briefly and place promptly on ice.
  3. Add:
    10X RT buffer 2 無
    RNAse inhibitor 1 無
    M-MuLV Reverse Transcriptase 1 無
    ______
    final volume 20 無
  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).

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