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RT-PCR

Reverse Transcriptase-PCR

This method was submitted by Jim Hutchins from the University of Mississippi Medical Center.
The following is a great RT-PCR protocol I obtained from David Klein at NIH. He gets all the credit; if you have problems with it, contact me :-)
Jim Hutchins
hutchins@umsmed.edu
http://www.umsmed.edu/~hutchins/

References

Protocol

PROCEDURE FOR cDNA SYNTHESIS ON DYNABEADS (5/4/94)
  1. Dissolve RNA (30 ug) in 10 ul H20, add 20 ul TE/1M KCl.
  2. a) Place 100 ul Dynabeads (5 mg/ml) in a 0.5 ml tube.
    b) Bind beads.
    c) Remove liquid.
    d) Add 100 ul TE/1M KCl.
    e) Wash.
    f) Bind beads.
    g) Remove liquid.
  3. a) Add RNA to beads.
    b) Heat to 70 C for 2 min and cool slowly to RT for 10 min.
    c) Bind beads.
    d) Remove liquid.
  4. Resuspend beads in :
    2.5 ul Buffer A* (200 mM Tris-HCl, pH 8.3,1.0 M KCl)
    2.5 ul Buffer B* (30 mM MgCl2 and 15 mM MnSO4)
    20.0 ul dNTPs (2.5 mM each)
    1.0 ul 32P-dCTP (5 uCi)
    1.0 ul RNasin-Pharmacia
    2.0 ul SuperScript II RT (200 U/ul)(Gibco BRL # 18064-014)
    5.0 ul Retrotherm RT (1 U/ul) (Epicentre Technologies # R19250)
    16.0 ul H20
    -----------------
    50 ul

    * These buffers are supplied with the Retrotherm RT.

  5. Remove 1 ul of reaction. This represents total 32P counts for use in calculating the amount of cDNA synthesized.
  6. Heat at 40 C for 30 min.
  7. Heat at 70 C for 1 hr.
  8. Bind beads and remove all liquid.
  9. Wash beads with 100 ul TE, bind beads, remove liquid.
  10. Resuspend beads in 100 ul TE. Count 1 ul of beads to calculate the amount of cDNA synthesized. Use 1 ul of beads per PCR.

    I think the real secret here is the removal of secondary structure by the thermostable RT coupled with the immobilized RNA forming a "reusable" pool of template. This works well with total RNA or a poly(A)+ fraction.

    Please contact me if there are any corrections to be made.

    Jim Hutchins
    hutchins@umsmed.edu
    http://www.umsmed.edu/~hutchins/


    Original method from:
    From: klein@helix.nih.gov (David Klein)
    Subject: Re: Fuzzy balls of mRNA