8 March 2002

Reagents:

  • 14C mevalonic acid DBED salt, NEC166, 0.1 µCi/µl

  • 1x PBS

  • H2O

  • Glass tubes, 16 ml, VWR 60828-387, 72/$134.02

  • Glass tubes, 50 ml, VWR 60828-423, 36/$112.25

  • Chloroform, 4 L, Stores

  • Methanol, 4 L, Stores

  • N2 gas stream

  • Petroleum ether (35-60°C), 1 L, Stores 0014-285, $8.99

  • Na2SO4 (anhydrous), 3 kg, Stores 0004-410, $28.09

  • Glass pasteur pipette

  • Glass wool

  • Toluene (purified), 4 L, Stores 0015-070, $17.45

  • Ethyl ether (anhydrous), 1 L, Stores 0012-625, $17.14

  • TLC silica gel 60 (20 x 20 cm), VWR EM-11845-7, 25/$231.53

  • Iodine (crystal), VWR MK098402, 125 g/$98.40

 

Protocol:

  1. Set up culture at 106 cells/ml in 100ml with 14C mevalonate
    (50 µl at 0.1 µCi/µl --> final 0.05 µCi/ml).

  2. Grow 0 to 48 hours.

  3. Wash with 1x PBS (important to remove FBS).

  4. Resuspend in 1ml H2O. Use glass tubes from here on.

  5. Add to 10ml chloroform:methanol (2:1 v/v).

  6. Stir at RT for 3 hours.

  7. Evaporate under N2 gas stream for ~1 hour until solution turns cloudy.

  8. Extract 2x with 10 ml petroleum ether at 40-60°C.

  9. Lower fraction ~500 µl, transfer upper fraction (x2) to 50 ml tube.

  10. Dry with anhydrous Na2SO4.

  11. Filter through pasteur pipette with glass wool.

  12. Evaporate to dryness under N2.

  13. Store at -20°C until use.

  14. Place 100 ml solvent in tank (toluene:diethylether 9:1 v/v).

  15. Resuspend samples in 100 µl petroleum ether (vaporizes rapidly).

  16. Spot at ~1.5 cm above bottom of gel.

  17. Place gel in tank and run about 1.5 hrs or until solvent front reaches about one inch from the top of the plate.

  18. Remove from tank and dry overnight to prevent 14C contamination of phosphor-screen.

  19. Cover in plastic wrap and expose on a phosphor-screen to visualize 14C-labeled compounds.

  20. To visualize markers, sprinkle crystals of iodine on the bottom of a sealable glass container.

  21. Place plates in container and seal lid.

  22. Wait 20-30 minutes for entire plate to be exposed.

  23. Circle visualized spots as staining will quickly fade.

  24. Calculate Rf value by dividing the distance traveled of substance by the distance traveled of solvent front.

Department of Microbiology
School of Medicine
University of Washington
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