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.
Organic waste: phenol, chloroform, and tubes or tips that have been in contact with them should be put in the plastic jars provided by Research Safety.
Materials
- 2-mL conical screw-cap tubes, with smooth caps
- 2-mL Eppendorf tubes
- 1-mm zirconium beads (for bacteria), baked at 200°C overnight in conical flask with metal spatula
- Bio101 Beadbeater
- 1X and 5X low-pH buffer (from 10X stock)
- 10X low-pH buffer
- 500 mM sodium acetate 41.0 g/L
- 100 mM EDTA 37.2 g/L
- adjust pH to 5.1 by adding concentrated HCl
- 20% (w/v) SDS
- Phenol, with 0.1% 8-hydroxyquinoline, pH 5.1
- Use amber bottle or clean bottle covered with aluminum foil.
- Warm 500 mL redistilled phenol in 60°C water bath.
- Keep bottle cap loose except when shaking.
- Add 0.5 g 8-hydroxyquinoline (antioxidant, makes phenol yellow).
- Add 500 mL 5X low-pH buffer, shake vigorously, allow phases to separate.
- Check pH of buffer with pH paper.
- Pipette off buffer, replace with 1X low-pH buffer, shake, check pH.
- Repeat until pH of buffer remains ~5.1.
- Store in the dark and keep refrigerated.
- I use phenol, saturated with buffer, pH 4.3 [Fisher: BP1751I-400] instead.
- Phenol: Chloroform: Isoamyl alcohol 25:24:1, pH 5.2 [Fisher: BP1753I-100]
- Chloroform: Isoamyl alcohol 24:1 [Sigma: C-0549]
- 5 M ammonium acetate
- 100% isopropanol (or 100% ethanol; advantage of isopropanol is that only 0.7 sample volume required, where as for ethanol, 2 sample volumes required)
- Ice bucket
Method
- 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 equilibrated phenol, pH 5.1
35 mL (w/v) SDS
300 mL 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 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.
Reference
Lin, C. and D. A. Stahl. 1995. Taxon-specific probes for the cellulolytic genus Fibrobacter reveal abundant and novel equine-associated populations. Appl. Environ. Microbiol. 61: 1348-1351.
MacGregor, B. J., D. P. Moser, E. W. Alm, K. H. Nealson, and D. A. Stahl. 1997. Crenarchaeota in Lake Michigan sediment. Appl. Environ. Microbiol. 63: 178-1181.
With contributions by Stefan Mützel, Kerstin Sahm, Laura Sappelsa, Richard Sharp, and Cherie Ziemer.