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Protocol for First-strand cDNA Synthesis
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Protocol for First-strand cDNA Synthesis
(PCR template generation with M-MuLV Reverse Transcriptase)

The following protocol is optimized to generate first-strand cDNA for use in two-step RT-PCR.
Mix and briefly centrifuge all components after thawing, keep on ice.

  1. Add into sterile, nuclease-free tube on ice in the indicated order:
    Template RNA total RNA
    or poly(A) RNA
    or specific RNA
    100 ng - 5 µg
    10 - 500 ng
    0.01 pg - 0.5 µg
    Primer oligo(dT)18
    or random hexamer
    or gene-specific
    0.5 µg (100 pmol)
    0.2 µg (100 pmol)
    15-20 pmol
      DEPC-treated Water to 12.5 µl
  2. Optional. If RNA template is GC rich or is known to contain secondary structures, mix briefly, centrifuge briefly and incubate at 65°C for 5 minutes, chill on ice, briefly centrifuge and place on ice.
  3. Add the following components in the indicated order:
    5X reaction buffer for Reverse Transcriptase 4 µl
    RiboLock™ RNase Inhibitor 0.5 µl (20 u)
    dNTP Mix, 10 mM each 2 µl (1 mM final concentration)
    M-MuLV Reverse Transcriptase 2 µl (40 u)
    Total volume: 20 µl

    Mix gently and centrifuge briefly.

  4. If oligo(dT)18 primer or gene-specific primer is used, incubate 60 minutes at 37°C.
    If  random hexamer primer is used, incubate 10 minutes at 25°C followed by 60 minutes at 37°C.
    For transcription of GC rich RNA reaction temperature can be increased to 45°C
  5. Terminate the reaction by heating at 70°C for 10 minutes. Do not heat-inactivate enzyme prior to analysis of long cDNA to avoid cleavage.

Note

Reference

  1. Sambrook, J., Russell, D.W., Molecular Cloning: A Laboratory Manual, the third edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2001.
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Updated kovo 18, 2008 11:05