By Derek Kinchington, Raymond F. Schinazi
Royal London college of medication, united kingdom. moment writer, Raymond F. Schinazi, is at Emory Univ., Decatur, GA. bargains uncomplicated and scientific researchers types to guage compounds potent opposed to acute and protracted infections. comprises entire assays, caliber trying out, and unpublished equipment. DNLM: Hepatitis, Viral, Human - drug treatment.
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Read the absorbance at 490 nm on a microplate reader to assess cell viability. 3. 1. Dot-Blotting Procedure 1. 1. and add 90 µL of 2X denaturation solution to each well, pipeting up and down several times to mix. 2. Incubate at room temperature for 20 min. 3. Cut the nylon membrane to size and prepare it for blotting by wetting it first with distilled water and then 20X SSC. 4. Dot-blot the denatured supernatants on to the nylon membrane as directed by the manufacturer of the blotting apparatus.
22-µ filters. All chemical and radioactive materials and waste are to be handled and discarded in accordance with each individual’s current institutional safety procedures. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. RPMI-1640 (without L-glutamine). L-Glutamine. Fetal bovine serum (FBS). Trypsin-versine solution. HEPES-buffered saline (HBS). G418 (Genticin™, Gibco-BRL, Gaithersburg, MD). 3TC (lamivudine, (–)β-L-2',3'-dideoxy-3'-thiacytidine).
7. Dulbecco’s phosphate-buffered saline (DPBS). 8. Trypsin:ethylenediaminetetra-acetic acid (EDTA) (Gibco-BRL Life Technologies, Gaithersburg, MD). 9. MTS (Owen’s reagent) solution: Dissolve 42 mg of MTS [3-(4,5-dimethylthiazol2-yl)-5-(3-carboxymethophenyl)-2-(4-sulfophenyl)-2H-tetrazolium] into 21 mL of Hep AD38 Assay 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 45 DPBS in a light protected container. 2-µm membrane. 92 mg/mL in DPBS. MTS/PMS Solution: Mix 100 µL of PMS solution with 2 µL of MTS solution just prior to use.