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Protocol

ISOLATION OF TOTAL RNA
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REAGENTS: 50 mM NaAc pH 5.2 20% SDS Ultrapure phenol-equilibrated w/ 5OmM NaAc pH 5.2 3M NaAc pH 5.2 PBS - phosphate buffered saline
Day 1: 1. Wash cells 3x with PBS 40C 2. Add 3ml of 5OmM NaAc pH 5.2, 1% SDS to cells. (3ml per 150mm dish or flask) 3. Scrape cells into a 15ml polypropylene tube containing 3ml of phenol pH 5.2 4. Vortex 5. Incubate at 600C for 15 min; agitate (vortex) frequently 6. Incubate on ice 151 7. Centrifuge 3,000 rpm 15min at OOC 8. Remove aqueous layer to 15ml (round bottom polyprop. tube) 9. Make aqueous layer 0. 3M Ka Ac and add 2 volumes cold ethanol (100%) 10. Incubate overnight -200C (or 2 hours dry ice/700C) Day 2: 11. Centrifuge -10,000 rpm in super speed Sorvol 40C 12. Wash pellet with 70% ETOH 13. Dry in speed vac 14. Resuspend in 200ul of lOmM TRIS pH 7.4 in a microfuge tube 5OmM NaCl 7mM MgCl2 15. Add DNase to 50ug/ml 16. 151 at RT 17. Add SDS to 0.5% 18. Phenol: CIAA extraction 19. CIAA extraction 20. Add NaAc to 0.3M > add 2 volumes EtOH 21. PPT overnight or as above 22. Microfuge 150 at high speed 23. Wash pellet with 70% EtOH 24. Dry pellet in speed vac 25. Resuspend in dH20
ISOLATION OF TOTAL RNA

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