0.12 M Sodium Phosphate Buffer (pH 7.2) (100 ml)
0.12 M Sodium Phosphate Buffer ---------- 12 ml of 1 M Sodium Phosphate Buffer
Deionized H2O ---------------------------- 88 ml
Proteinase K (1 ml)
0.5 mg Proteinase K ----------------------- 25 ul of Proteinase K (20 mg/ml)
Deionized H2O ---------------------------- 975 ul
Store at -20oC.
6 M Guanidine thiocyanate (100 ml)
Guanidine thiocyanate ---------------------------- 70.9 g
Add deionized H2O to make a final volume of ---- 100 ml
Store at 4oC.
Acid phenol: chloroform: IAA (25: 24: 1)
PROCEDURES
All manipulations should be performed on ice! Always wear gloves!
1. Inoculate bacteria into 10 ml of LB and grow overnight at 37oC.
2. Use 5 ml of this culture to inoculate 50 ml of LB and grow for 2.5 hours at 37oC, to an absorbance A600 of 0.5-0.6.
3. Centrifuge a 1.5 ml culture in a microfuge tube at 13,000g for 2 minutes.
4. Resuspend the pellet in 300 ul of 0.85% NaCl.
5. Centrifuge at 13,000g for 2 minutes and discard the supernatant.
6. Resuspend the pellet in 400 ul of 0.12 M Sodium Phosphate Buffer
(pH7.2).
7. Add 50 ul of 5% SDS and 50 ul of Proteinase K (0.5 mg/ ml).
8. Vortex and incubate at 37oC for 20 minutes.
9. Add 750 ul of 6 M Guanidine thiocyanate.
10. Vortex and centrifuge at 13,000g for 3 minutes.
11. Transfer the supernatant to a fresh tube.
12. Extract sample with Acid phenol: chloroform: IAA.
13. Add 0.1 volume of 3 M Sodium acetate (pH 7.0) and 2.5 volume of cold ethanol.
14. Precipitate RNA at -20oC for 1 hour.
15. Centrifuge at 13,000g for 20 minutes and discard the supernatant.
16. Wash the pellet with 70% ethanol by resuspending and centrifuging
at 13,000g for 10 minutes.