Reverse Transcriptase, Recombinant HIV Assay

Unit definition: One unit incorporates 1 nanomole of tritiated dTMP into acid insoluble productsusing poly(A)•oligo(dT) 12-18 as the template-primer in 20 minutes at 37° C.

Reagents

  • 0.05 M Tris, pH 8.3, containing 0.008 M MgCl2
  • 1 mg/ml polyadenylic acid in water (poly A)
  • DNA primer:Oligo d(T)12-18
  • 1 μ mole dTTP/mL stock solution
  • [methyl-3H]-Thymidine 5'-triphosphate (3H-dTTP)
  • dTTP-3H-dTTP working mix: Add 1-2 μL 3H-dTTP per mL of 100 nmol/mL dTTP in order to obtain 1 to 1.5 x 105 cpm/mL
  • 1% bovine serum albumin
  • 10% perchloric acid
  • 1% perchloric acid
  • Buffer substrate reaction mixture: Prepare fresh, immediately before use:
    For each 1mL of reaction mixture required mix:
    0.7 mL Tris/HCl, pH 8.3, 0.008M MgCl2
    0.3 mL 1 mg/mL poly(A) RNA template
    0.005 mL 0.02 mg/mL oligo d(T)12-18 DNA primer
    0.02mL 1% BSA

Enzyme: dilute as needed wtih 0.05M Tris/HCl, pH 8.3, 0.008M MgCl2 containing 0.1 mg/mL (1%) BSA

Procedure

Pipette into each tube as follows:

Buffer substrate mix 0.1 mL
dTTP-3H3-dTTP 0.1 mL
Enzyme 5-10 μL

Incubate 20 minutes at 37° C. Stop reaction by adding 1 ml 10% cold perchloric acid. Filter through 0.2μ manifold filters used with Millipore vacuum manifold. Wash four times using 2mL 1% cold perchloric acid/wash. Transfer filter to scintillation vials. Add 2mL Cellosolve (or 2-methoxyethanol) to dissolve filter. Filters become opaque upon addition of Cellosolve. Make sure filters are dissolved before proceeding. Add 10mL scintillation cocktail and count.

Calculations

calculations

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