From Tissue:
1. In a sterile14 ml tube add 100-500 mg of tissue.
2. Add 1ml Trizol per 100 mg of tissue.
3. Clean homogenizer 3X with 5ml DEPC-treated H2O.
4. Homogenize samples 2X/10 seconds/setting=5.
5. Incubate 5 minutes/RT to let debris settle, or spin down at 	2,000rpm/5 min./4'C.
6. Aliquot 1 ml of supernatant into a 1.5 ml tube.
Proceed to step seven.

From dishes:
1. Add 1 ml Trizol to each well if using six well plates.
2. Shake for 3-4 minutes with lid on.
3. Pipet Trizol from dish into clean, 1.5 ml microfuge tubes.
Proceed to step seven.

7. Add 0.2 ml chloroform/ml trizol, cap then vortex for 15 seconds.
8. Incubate RT/up 2-3 minutes to allow phase separation.
9. Spin at 12,000.rpm/15 minutes/4'C.
10. Remove the upper, aqueous layer (600ul). Add to a new1.5ml 	tube.
11. Add 0.5 ml IPA per ml Trizol. Mix well, incubate RT/5-10 minutes. 

 	Or at -20'C for 2 hours for low RNA concentrations.
12. Spin at 12,000.rpm/30minutes/4'C. Note pellet.
13. Aspirate supernatant.
14. Wash 1x with 1.0ml of 75% ETOH (4'C) in DEPC H2O per ml Trizol.
15. Vortex, then spin down at 7,500.rpm/5 minutes/4'C.
16. Aspirate supernatant then spin down in small speed vac to 	
	remove ETOH. Do not overdry!
17. Dissolve in 50-100 ul DEPC treated H2O or 0.5% SDS in DEPC 	water.
18. Incubate at 65'C for 10 minutes, mix then spin down.
19. Remove 2 ul and add to 100 ul H2O for A260/280.
      ug/ul= A260 x 2.0 if as above. If 1 ul/100 ul H2O multiply by 4.