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HIV reverse transcriptase activity assay

1. CEM-SS cells (~1250 cells per well) are added to 96-well plates containing drugs.

2. Cells are infected with HIV at the multiplicity of 0.005-2.5.

3. Reverse transcriptase activity is assayed 6 days after infection.

4. Prepare fresh RT reaction buffer :

1M EGTA 125 ml
dH2O 125 ml
Triton X-100 125 ml
1M Tris-HCl (pH 7.4) 50 ml
1M DTT 50 ml
1M MgCl2 40 ml

5. Mix 1 part 3H-TTP (5 Ci/ml), 2.5 part Poly rA/oligo dT, 2.5 parts RT reaction buffer and 4 parts distilled water.

6. Add 10 ml reaction mix and 15 ml of virus containing supernatant to each well of 96-well round bottom microtiter plate.

7. Incubate at 37 C for 60 minutes.

8. Spot the reaction (25 ml) onto filter mats.

9. Wash 6 times (5 minutes each) in a 5% sodium phosphate buffer.

10. Wash 2 times (1 minute each) in distilled water, 2 times (1 minute each) in 70% ethanol.

11. Dry.

12. Place the dried filter mat in a plastic sample bag.

13. Add betaplate scintillation fluid and heat-seal the bag.

14. Read using a Wallac Microbeta scintillation counter.