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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 l (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 37C.
    If  random hexamer primer is used, incubate 10 minutes at 25C followed by 60 minutes at 37C.
    For transcription of GC rich RNA reaction temperature can be increased to 45C
  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