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.

Department of Microbiology
School of Medicine
University of Washington
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