Please find the following protocol:

Reverse Transcriptase Assay Optimized for Avian Sarcoma Virus


Overview:

This protocol assays for reverse transcriptase activity by monitoring the incorporation of [3H]-dGTP into the DNA strand primed by the oligonucleotide dG primer and synthesized off a poly-C RNA template.


Procedure:

1. Clarify 2 to 10 ml of virally infected cell culture supernatant in a JA20 rotor at 10,000 rpm (7,500 X g) for 10 min.

2. Recover the supernatant and centrifuge in a SW41 rotor for 45 min at 36,000 rpm (160,000 X g) to pellet the virus. Top off the centrifuge tube with media.

3. Drain the centrifuge tube well and keep the tube on ice while resuspending the virus pellet in 100 μl of Viral Dilution Buffer. Pipette the solution up and down a few times and then vortex for a few seconds to resuspend. Avoid introducing air bubbles.

4. Combine 20 μl of the resuspended virus and 20 μl of Reaction Mixture. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.

5. Incubate samples at 37°C for 0 min (for a negative control), 120 min, and overnight.

6. Stop the reaction by the addition of Sodium Pyrophosphate to 0.1 M final concentration.

7. Add 80 μg of Salmon Sperm DNA as a carrier and 1 ml of ice-cold 10% TCA.

8. Incubate on ice for 10 min and load the precipitate onto glass fiber filters on a vacuum manifold.

9. Rinse the fibers extensively with cold NaP-P/HCl and then once with 95% Ethanol.

10. Dry the fibers and count in a scintillation counter.


Solutions:

95% (v/v) Ethanol
NaP-P/HCl (2 liters)............90 g Sodium Pyrophosphate (NaP-P)
.........Add ddH2O to a final volume of 2 liters
................................................120 ml HCl
10% Trichloroacetic Acid (TCA)...10% (w/v) Trichloroacetic Acid
Reaction Mixture......................................0.1 μl/μCi [3H]-dGTP
...........................................0.1% (v/v) NP-40
.............................2 mM DTT (add before use)
...................600-900 μg/ml Oligonucleotide dG
..............................10 mM Magnesium Acetate
...................................50 mM Tris-HCl, pH 8.1
...........................1.25 μg/μl Polynucleotide-rC
Viral Dilution Buffer
.......100 μg/ml recrystallized Bovine Serum Albumin
............................... 10 mM DTT (add before use)
........................................100 mM Tris-HCl, pH 8.1
.....................................10 mM Magnesium Acetate


Bioreagents and chemicals:

Tris-HCl
Magnesium Acetate
[3H]-dGTP
Oligonucleotide
Polynucleotide-rC
Oligonucleotide
DNA, Salmon Sperm
NP-40
Bovine Serum Albumin (BSA)
Trichloroacetic Acid (TCA)
Sodium Pyrophosphate
Hydrochloric Acid
Ethanol
DTT

Reference Link:

http://www.bio.com/protocolstools/protocol.jhtml?id=p615