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First-Strand cDNA Synthesis Using M-MLV RT

  • Researcher: Chang Ko-Yu, Chu Yi-Chia
  • Place: Academia Sinica
  • Date: April 30th, 2015

Material

  1. randon primer 1㎕
  2. 2MUG RNA F10 1.95㎕
  3. 2MUG RNA m1 1.95㎕
  4. 10mM dNTP 1㎕
  5. ddH2O
  6. 5× FSB 4㎕
  7. 0.1M DTT 2㎕
  8. RNase out 1㎕
  9. M-MLV RT 1㎕
  10. eppendorf

Procedure

A 20-㎕ reaction volume can be used for 1 ng-5MUG of total RNA or 1-500 ng of mRNA.

  1. Add the following components to a nuclease-free microcentrifuge tube:
    • 1㎕ oligo(dT)12-18(500μg/ml), or 50-250 ng random primers, or 2pmole gene -specific primer
    • 1 ng to 5MUG total RNA or 1 ng to 500ng of mRNA
    • 1㎕ 10 mM dNTP Mix(10 mM each dATP,dGTP,dCTP and dTTP at neutral pH)
    • Sterile, distilled water to 12㎕
  2. Heat mixture to 65℃ for 5 min an quick chill on ice. Collect the contents of the tube by brief centrifugation and add:
    • 4㎕ 5X First-Strand Buffer
    • 2㎕ 0.1 M DTT
    • 1㎕ RNaseOUTTM Recombinant Ribonuclease Inhibitor (40 units/㎕)

    (Note: When using less than 50 ng of starting RNA, the addition of RNaseOUTTM is essential.)

  3. Mix contents of the tube gently and incubate at 37℃ for 2 min.
  4. Add 1㎕(200 units) of M-MLV RT,a and mix by pipetting gently up and down. If using random primers, incubate tube at 25℃ for 10 min.
  5. Incubate 50 min at 37℃.
  6. Inactivate the reaction by heating at 70℃ for 15 min.

Result

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