Molecular markers
DNA purification
Organic extraction

Author: Rodney E. Shackelford, D.O., Ph.D. (see Authors page)

Revised: 29 May 2018, last major update April 2012

Copyright: (c) 2008-2018, PathologyOutlines.com, Inc.

PubMed Search: Organic extraction[TIAB] DNA purification

Cite this page: Shackelford, R.E. Organic extraction. PathologyOutlines.com website. http://www.pathologyoutlines.com/topic/moleculardnapurorganicextract.html. Accessed July 17th, 2018.
Technique
  • Mix crude cell preparations in a denaturing aqueous solution (often guanidinium thiocyanate or 2-mercaptoethanol) with a roughly equal volume of a phenol-chloroform solution and centrifuge the sample
  • Isoamyl alcohol may be added to phenol-chloroform solution to reduce foaming during extraction
  • Following vigorous mixing, the denatured proteins and lipids partition into the lower organic phase or at the aqueous / organic phase interface, while the nucleic acids partition into the upper aqueous phase allowing separation
  • Aqueous sample is often extracted using phenol-chloroform several times for greater nucleic acid purity
  • Alternatively, the aqueous solution is washed several times with an isoamyl alcohol-chloroform solution to remove residual phenol
  • Nucleic acids are often further purified by ethanol precipitation
  • Must use polypropylene tubes, as phenol-chloroform dissolves polystyrene tubes
Disadvantages
  • Phenol is toxic and can cause severe chemical burns, so protection (gloves, safety glasses, lab coat) and a fume hood are required
  • pH of solution must be correct (pH ~7.5), as DNA will partition into organic phase with pH ~5
  • Relatively cumbersome and slow compared to other recent organic extraction methods
  • Advantage: relatively long nucleic acid polymers can be extracted, useful for some research and molecular diagnostic applications
Diagrams / tables

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Phenol-chloroform extraction