Reverse Transcriptase Assay Optimized for Molonoy Murine Leukemia Virus (MuLV)


Overveiw:

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


Protocol:

1. Clarify 5 ml of virally infected cell culture media by centrifugation in a JA20 rotor at 7,000 rpm (4,000 X g) for 10 min.

2. Take the supernatant and pellet the virus by centrifugation in a SW41 rotor at 35,000 rpm (150,000 X g) for 1 hr. Top off centrifuge tube with Tris-Glucose.

3. Drain the pellet well. Keeping the tube on ice, suspend the virus pellet in 100 μl of Viral Dilution Buffer.

4. Scrape the pellet with a pipettor up and down and avoid introducing bubbles.

5. Combine 20 μl of the resuspended virus and 20 μl of Reaction Mixture.

6. Incubate overnight at 33°C.

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

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

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

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

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


Solutions:

95% (v/v) Ethanol
NaP-P/HCl.....................................90 g Sodium Pyrophosphate
......................................................ddH2O to 2 liters
..............................................................120 ml HCl
10% (w/v) Trichloroacetic Acid (TCA)
Reaction Mixture........0.1 μl/μCi [3H]-dGTP CAUTION! Biohazard
....................................................60 mM NaCl
..............................0.1 μg/μl Polynucleotide-rC
.................................................For 34 assays
........................70 μl of [3H]-dGTP at 1 mCi/ml
...........................................8.4 μl of 5 M NaCl
..................................2.8 μl of 5% (v/v) NP-40
.............................................4.9 μl of 1 M DTT
.............................10 μg/ml Oligonucleotide dG
.............................35 μl of 1 M Tris-HCl, pH 8.1
..........................................0.02% (v/v) NP-40
..............................7 mM DTT (add before use)
...................7 μl of 10 mg/ml Polynucleotide rC
...........35 μl of Oligonucleotide-dG at 200 μg/ml
final concentrations in the Reaction Mixture:
...................467 μl of distilled, deionized ddH2O
....................................50 mM Tris-HCl, pH 8.1
.....................70 μl of 0.1 M Magnesium Acetate
...............................10 mM Magnesium Acetate
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


Bioreagnets and Chemicals:

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


Reference Link:

http://www.bio.com/protocolstools/protocol.jhtml?id=p618