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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- 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.
- Add: 500 mL μL equilibrated phenol, pH 5.1
35 mL μL (w/v) SDS
300 mL μL 5X low-pH buffer
- 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.)
- Bead beat at speed 5 for 30 sec. Incubate on ice for 1 min. Bead beat again at speed 5 for 30 sec.
- Centrifuge at 14,000 rpm for 5 min at 4°C to separate phases.
- Transfer the top aqueous phase to fresh Eppendorf tube.
- Add 100 mL μL 1X low-pH buffer to the remaining sample.
- Bead beat, as above.
- Centrifuge, combine top aqueous phase with the first one.
- 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.)
- 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.
- Add equal volume of chloroform. Vortex and centrifuge.
- Transfer final aqueous phase to fresh tube.
- 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.
- Centrifuge for 30 min at 4°C.
- Carefully decant isopropanol.
- Wash with 80% (v/v) ethanol.
- Centrifuge to collect the pellet.
- Dry pellet using vacuum or air dry.
- Resuspend pellet in RNase-free water.
- Store at -80°C.
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