Reverse Transcriptase PCR

Procedure for cDNA Synthesis on Dynabeads

  1. Dissolve RNA (30 ug) in 10 ul H20, add 20 ul TE/1 M KCl.

  2. Preparation of Dynabeads:
    1. Place 100 ul Dynabeads (5 mg/ml) in a 0.5 ml tube.

    2. Bind beads to side of tube with magnet.

    3. Aspirate off supernatant.

    4. Add 100 ul TE/1M KCl and mix with beads.

    5. Bind beads to side of tube with magnet.

    6. Aspirate off supernatant.

  3. Add RNA to beads.

  4. Heat to 70oC for 2 minutes, then cool slowly to room temperature for 10 minutes.

  5. Bind beads to side of tube with magnet.

  6. Aspirate off supernatant.

  7. 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, 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)
    • 5.0 ul Retrotherm RT (1 U/ul) (Epicentre Technologies)
    • 16.0 ul H20
    * These buffers are supplied with the Retrotherm RT.

  8. Remove 1 ul of reaction and count in scintillation counter. This represents total 32P counts for use in calculating the amount of cDNA synthesized.

  9. Heat remaining reaction at 40oC for 30 minutes.

  10. Heat remaining reaction at 70oC for 1 hr.

  11. Bind beads to side of tube with magnet.

  12. Aspirate off supernatant.

  13. Add 100 ul TE and mix beads thoroughly.

  14. Bind beads to side of tube with magnet.

  15. Aspirate off supernatant.

  16. Resuspend beads in 100 ul TE.

  17. Count 1 ul of beads in scintillation counter to calculate the amount of cDNA synthesized. Use 1 ul of beads per PCR.

PCR

  1. Set-up 100 ul PCR reactions:

    • 79 ul distilled H2O
    • 1 ul 1 M Tris, pH8.5
    • 1.5 ul 3 M KCl
    • 5 ul 20% Triton X-100
    • 2 ul 10 mM dNTPs
    • 6 ul 25 mM MgCl2
    • 2 pmoles primer1, usually 2 ul of a 100 pM stock
    • 2 pmoles primer2, usually 2 ul of a 100 pM stock
    • 1 ul Dynabeads with cDNA
    • 0.5 ml Taq DNA Polymerase (optional)

    Note: Volume of distilled H2O can be adjusted if additional volumes of reagents are required.


  2. PCR Cycles (for PE 9600 or equivalent):

    95oC for 5 minutes

    95oC for 30 seconds
    55oC for 1 minute
    72oC for 2 minutes
      ==> 35 cycles

    72oC for 10 minutes
    4oC hold

    Note: The incubation times at each temperature can be varied to accomodate different volumes of reactions.

    Also, annealing temperature may have to be determined emperically.