TRIZOL Method to Isolate RNA and Protein
From Invitrogen Life Technologies
1. Homogenize (in the fume hood with goggles) approximately 0.1mg tissue (about
1/3 size of adult mouse heart) in 1ml Trizol and place in eppindorf tube
2. Sit for 5 minutes at room temperature
3. add 0.2ml chloroform, mix by inversion and sit for 2 minutes
4. centrifuge for 15 minutes at 12000g or 11.4 rpm
RNA:
5. transfer upper (clear) layer to fresh eppindorf tube
6. add 0.5ml of isopropyl alcohol (2-propanol) and sit for 5 minutes
7. spin at 12000g (11.4rpm) for 10 minutes
8. remove supernatant and wash 1 times in 75% ethanol
9. spin at 7500g for 5 minutes
10. dry pellet (for 1-2 hours) and re-suspend in 50ul DEPC treated H2O
Protein:
5. keep lower (pink) layer and interphase in the tube
6. add 0.3ml of 100% ethanol and sit for 2-3 minutes (will get very goopy)
7. spin for 5 minutes at 2000g (4600rpm)
8. remove half of the supernatant (still pink) into a fresh tube (throw out pellet)
9. add 750ul of isopropyl alcohol (2-propanol) and sit for 10 minutes
10. spin at 12000g (11.4 rpm) for 10 minutes
11. remove supernatant and wash pellet ( vigorously homogenize) with 1ml of 0.3M
guanidine HCl in 95% ethanol
12. spin at 7500g (8900 rpm) for 5 minutes
13. repeat steps 11-12 3 times over 20 minutes
14. vortex in 1 ml of ethanol and sit for 20 minutes
15. spin at 7500g (8900rpm) for 5 minutes
16. dry pellet (1-2 hours)
17. re-suspend in an appropriate volume of NewRipa (plus inhibitors)
18. spin for 10 minutes at max (13.4 rpm) and collect supernatant (protein)