Reagents:

  • Guanidine Thiocyanate (Fluka, FW=118.2, 250 g, 50990, $77.15)

  • Citric Acid (Sigma, FW=294.1, 500 g, C-8532, $14.25)

  • Sarkosyl (Sigma, FW=293.4, 50 g, L-9150, $14.75)

  • 2-β Mercaptoethanol (Sigma, FW=78.13, 100 ml, M-7522, $15.40)

  • RNase free ddH2O (Sigma, 1 L, W-4502, $23.55)

  • 3M Sodium Acetate Buffer (pH 5.2) (Sigma, 100 ml, S-7899, $11.90)

  • Phenol (Boehringer Mannheim, 500 g, 1814303, $88.00)

  • Chloroform (UW Stores, 4 L, 0011-555, $36.14)

  • Isoamyl Alcohol (Sigma, FW=88.15, 500 ml, I-9392, $22.65)

  • Isopropanol (UW Stores, 4 L, 0014-620, $22.08)

  • 75% and 100% Ethanol

  • 1 M Tris-HCl (pH 7.6) (Sigma, 1L, T-2788, $0.00)

  • 20cc Syringe (UW Stores, 40/box, 0056-590, $11.12)

  • 18Gx1-1/2 Needle (UW Stores, 100/pack, 0053-560, $4.05)

Solution D

Final Concentration

Amount (100 ml)

Guanidine thiocyanate

4 M

47.26 g

Citric Acid (1 M, pH 7.0)

25 mM

2.5 ml

Sarkosyl (10%)

0.5%

5 mL

2-β Mercaptoethanol (14 M)

0.1 M

0.72 ml*

RNase free ddH2O

 

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.

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


Protocol:

 

1. Lyse cells in solution D (200 µl/l X 106 cells).

2. Homogenize by passing lysate five times through 18-gauge needle with syringe. Store at �70 °C or continue.

3. Sequentially add the following:

                0.1 volume         3 M Sodium Acetate (pH 5.2), mix

                1.0 volume         Phenol, water-saturated, mix

                0.2 volume         Chloroform/Isoamyl Alcohol (24:1) (fresh)

4. Vortex 10 seconds.

5. Incubate on ice 15 minutes.

6. Centrifuge 30 minutes at 10,000x g (brake slowly), 4°C.

7. Transfer upper aqueous phase to new tube.

8. Precipitate with an equal volume of Isopropanol for 30-60 minutes, -20°C.

9. Centrifuge 30 minutes at 10,000x g (brake max), 4°C.

10.  Dissolve pellet in 600 µl solution D.

11.  Transfer to Eppendorf tube.

12.  Sequentially add the following:

                  60 µl        3 M Sodium Acetate (pH 5.2), mix

                600 µl        Phenol, water-saturated, mix

                120 µl        Chloroform/Isoamyl Alcohol (24:1) (fresh)

13.  Vortex 10 seconds.

14.  Incubate on ice 15 minutes.

15.  Centrifuge 15 minutes at 14,000 rpm.

16.  Transfer upper aqueous phase to new tube.

17.  Precipitate with an equal volume of Isopropanol for 30-60 minutes, -20°C.

18.  Centrifuge 5 minutes at 14,000 rpm.

19.  Wash pellet with 200 µl 75% Ethanol.

20.  Centrifuge 1 minute at 14,000 rpm.

21.  Air dry and resuspend in 500 µl RNase-free ddH2O.

22.  Make 1:100 dilutions in 10mM Tris pH 7.6 to read OD260/280.

23.  Run 100-500 ng total RNA on Bioanalyzer.

24.  Make 250 µg aliquots.

25.  Store at �70 °C with 0.1 volumes 3M Sodium Acetate (pH 5.2) and 2.5 volumes 100% Ethanol.


Notes:

- Phenol is a poison and causes burns. Use gloves and eye protection.

- Carry out all steps at room temperature unless otherwise stated.

- The procedure can be carried out in sterile, disposable, 50 ml conical tubes (Falcon, Greiner).

- Do not let the RNA pellet dry completely as this greatly decreases its solubility.

- Dissolve the pellet in DEPC-treated water. Incubate 10 to 15 minutes at 55-60°C. Expected yield: 50 to 80 µg for 10 x 106 cells.

- Water used for spectrophotometric measurement of RNA should have pH > 7.5. Typically, distilled water has pH < 6. Adjust water to a slightly alkaline pH by adding concentrated Na2HPO4 solution to a final concentration of 1 mM.

 

Solution D:

To Guanidine Thiocyanate bottle (250 g) add 293 ml ddH2O preheated to 60°C, and 13.25 ml 1M Citric Acid (pH 7.0). Keep at 60°C and mix until dissolved. Add 26.5 ml of 10% (w/v) N-lauroylsarcosine (Sarkosyl) and water to 530 ml total volume. For use add 324 µl 2-β mercaptoethanol (2-ME) to 45ml solution.

 

Sodium acetate, 2M:

Add 16.42 g sodium acetate (anhydrous) to 40 ml water and 35 ml glacial acetic acid. Adjust solution to pH 4.0 with glacial acetic acid and dilute to 100 ml final with water (solution is 2 M with respect to sodium ions). Store up to 1 year at room temperature.

 

Water-saturated phenol:

Dissolve 50 g phenol in 50ml ddH20 at 60 to 65°C. Aliquot in 50 ml conical tubes wrapped in aluminium foil and store at �20°C. Do NOT use buffered phenol in place of water-saturated phenol.

Department of Microbiology
School of Medicine
University of Washington
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