Reagents:

• Guanidine Thiocyanate (Fluka)

• 1 M Sodium Citrate (pH 7.0)

• 10% Sarcosyl

• 14 M 2-β Mercaptoethanol

• Rnase free H₂O

• 2 M Sodium Acetate (pH 5.2)

• water saturated Phenol

• 49:1 Chloroform/Isoamylalcohol

• Isopropanol

• 75% Ethanol

• DNase I (Promega)

• 10x reaction buffer (400 mM Tris-HCl, pH 7.9; 60 mM MgCl₂; 100 mM NaCl; 100 mM CaCl₂; 1 mM DTT)

Solutions

Solution D	Final Conc.	Amount (100 ml)
Guanidine thiocyanate	4 M	47.25 g
Sodium Citrate (1 M)	25 mM	2.5 mL
Sarcosyl (10%)	0.5%	5 mL
2-β Mercaptoethanol (14 M)	0.1 M	0.72 ml*
RNase free H2O		to 100 mL

* β-Mercaptoethanol should be added to the amount of solution D that is to be used that day. Make up solution D without β-Mercaptoethanol and then add 7.2 µl/ml of solution D as needed. Solution D without β-Mercaptoethanol can be stored at room temperature for 1 month.

Protocol:

1. Lyse cells in solution D (100 µl/1 X 10⁶ cells).

2. Sequentially add the following:

   0.1 Vol 2M Sodium Acetate, mix

   1 Vol Phenol

   0.2 Vol Chloroform/Isoamylalcohol

3. Vortex 10 seconds.

4. Incubate on ice 15 minutes.

5. Centrifuge 30 minutes at 10K, 4°C.

6. Transfer aqueous (top) phase to new tube.

7. Precipitate with 1 volume Isopropanol at least 1 hour, -20°C.

8. Centrifuge 30 minutes at 10K, 4°C

9. Dissolve pellet in 300µl solution D.

10. Transfer to Eppendorf tube.

11. Precipitate with 1 volume Isopropanol at least 30 minutes, -20°C.

12. Centrifuge 30 minutes at 14K in Eppendorf tabletop centrifuge.

13. Wash pellet with 75% Ethanol.

14. Centrifuge 10 minutes at 14K.

15. Dry and resuspend in appropriate RNAse free reagent (ddH₂O, poly A selection buffer).

16. Read O.D. 260/280.

17. Set up DNase I reaction in 1x buffer (10 U/250 µg RNA in 200 µl).

18. Incubate at 37°C for 1 hour.

19. Sequentially add the following:

    0.1 Vol 2 M Sodium Acetate, mix

    1 Vol Phenol

    0.2 Vol Chloroform/Isoamylalcohol

20. Vortex and centrifuge 15 minutes at 14K.

21. Remove aqueous (top) phase to new tube.

22. Precipitate with 1 volume Isopropanol at least 30 minutes, -20°C.

23. Centrifuge 30 minutes at 14K.

24. Wash pellet with 75% Ethanol.

25. Centrifuge 10 minutes at 14K.

26. Dry and resuspend in appropriate RNase free agent.

27. Read O.D. 260/280 and calculate % yield.

28. Aliquot, store at �70°C under alcohol.

Chomczynske, P. and Sacchi, N., Analytical Biochem. 162: 156-159, 1987.