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Molecular Pathology

DNA purification

Organic Extraction


Author: Rodney E. Shackelford, D.O., Ph.D. (see Reviewers page)
Revised: 14 April 2012, last major update April 2012
Copyright: (c) 2008-2011, PathologyOutlines.com, Inc.

Technique
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● 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
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● 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
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Phenol-chloroform extraction

Additional references
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Free training course-US Dept of Justice

End of Molecular Pathology > DNA Purification > Organic Extraction


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