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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.
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