RNA Isolation II (son of RNA)
FOR SMALL AMOUNTS OF TISSUE
For this procedure, you will need the following:
Chaos Buffer *
2M NaAc, PH 4.0 *
Phenol
Chloroform Iso-Amyl Alcohol (C.I.A.)
Isopropyl Alcohol
70 % Ethanol *
dH2O *
Must be RNase Free
1. Homogenize tissue in 400 ul of Chaos buffer, (use glass-glass tissue grinder)
* Note - Mix thoroughly after this and next 4 steps
2. Add 40 ul 2M NaAc PH 4.0. Mix by vortexing.
3. Add 400 ul of Phenol. Mix well, then add 80ul of C.I.A., mix again.
4. Let sit on ice for 10 minutes.
5. Microfuge for 20 minutes, keep supernatant.
6. Add 1 volume of Isopropanol to supernatant (should be around 500ul). Vortex,and mix by inverting.
7. Store for 30 minutes at -20c.
8. Spin for another 20 minutes.
9. Bring pellet up in 300 ul of Chaos buffer, vortex into solution.
10. Add 300 ul of Isopropanol. Chill on ice for 10 minutes.
11. Microfuge for 30 minutes
12. Resuspend pellet in 70% ethanol. Let sit for 15 minutes at room temperature.
13. Spin for 15 minutes.
14. Resuspend pellet in 200 ul of RNase free dH2O
15. Add 20 ul of 10x (150mM Tris, ph 7.6-7.8, 100mM MgCl, 100mM NaCl, 50mMDTT)
2 ul RNasin (RNase inhibitor)
2 ul of RNase Free DNase.
16. 0.5 hour 37oC
17. Extractions: Phenol, Phenol-CIA, CIA; EtOH precipitate and dry down in a speed vac.
18. bring up in 175ul. Take 25 ul to determine O.D.
Chaos Buffer for RNA isolation: For 100 mls 200 mls
4.5M Guanidinium thiocyanate 53.2 g 106.4
2% N-lauroylsarcosine (sarcosyl) 2.0g 4.0g
50mM EDTA 0.5M 10 mls 20 mls
25mM Tris-HCl, pH 7.5 1M 2.5 mls 5 mls
0.1M b-mercaptoethanol 700ul 1.4mls
0.2% antifoam A (Sigma) 200 ul 400 ul