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Contact: Slil Siripong

N.B. All solutions must be made with RNase-free water. Glassware has to be baked at 180-200°C for 8 hours. Plasticware should be unopened. Gloves should be worn all the time, and changed frequently.

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1. For each sample (~0.2 g), weight 0.5 g zirconium beads into a 2-mL screw-cap tube. For most sediments, need at least 8 tubes per sample.
2. Add:    500 mL μL equilibrated phenol, pH 5.1

               35 mL μL (w/v) SDS

               300 mL μL 5X low-pH buffer

3. Label four 2-mL Eppendorf tubes per sample – one each for phenol extraction, phenol-chloroform extraction, chloroform extraction, and isopropanol precipitation. (The isopropanol tube will be for storage, so label it completely.)
4. Bead beat at speed 5 for 30 sec. Incubate on ice for 1 min. Bead beat again at speed 5 for 30 sec.
   1. Centrifuge at 14,000 rpm for 5 min at 4°C to separate phases.
   2. Transfer the top aqueous phase to fresh Eppendorf tube.
   3. Add 100 mL μL 1X low-pH buffer to the remaining sample.
   4. Bead beat, as above.
   5. Centrifuge, combine top aqueous phase with the first one.
5. Add equal volume of phenol, pH 5.1 to the sample, vortex 20 sec, centrifuge for 5 min. Transfer aqueous phase to fresh Eppendorf tube without disturbing the interface. (N.B. If there is a visible layer at the organic/aqueous interface following phenol, phenol-chloroform, and/or chloroform extraction, repeat the extraction until the interface is clear.)
6. Add equal volume of Phenol: Chloroform: Isoamyl alcohol 25:24:1, pH 5.2 . Vortex and centrifuge as before. Transfer aqueous phase to fresh Eppendorf tube.
7. Add equal volume of chloroform. Vortex and centrifuge.
8. Transfer final aqueous phase to fresh tube.
9. Add:    7.5 M NH₄Ac to 2.5 M (i.e., 0.5 volume sample)

               0.7 volume isopropanol

               [MgCl₂ to 2 mM may help with low RNA samples]

               Vortex to mix. Incubate at -20°C overnight.

10. Centrifuge for 30 min at 4°C.
    1. Carefully decant isopropanol.
    2. Wash with 80% (v/v) ethanol.
    3. Centrifuge to collect the pellet.
    4. Dry pellet using vacuum or air dry.
    5. Resuspend pellet in RNase-free water.
    6. Store at -80°C.

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