Phenol/chloroform extraction

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Extract the sample two times with an equal volume of chloroform:isoamyl alcohol to remove any trace phenol.
Extract the sample two times with an equal volume of chloroform:isoamyl alcohol to remove any trace phenol.
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Precipitate the nucleic acid.
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Precipitate the nucleic acid. ([[nucleic acid precipitation]])
==Reagents==
==Reagents==
*Phenol equilibrated to pH 7.5
*Phenol equilibrated to pH 7.5
*Chloroform:isoamyl alcohol in a 24:1 ratio (CHIZAM! in [http://www.scripps.edu/mb/joyce/ Joyce lab] lingo)
*Chloroform:isoamyl alcohol in a 24:1 ratio (CHIZAM! in [http://www.scripps.edu/mb/joyce/ Joyce lab] lingo)

Revision as of 23:03, 14 July 2005

General Information

Phenol/chloroform extraction is an easy way to remove proteins from your nucleic acid samples and can be carried out in a manner that is very close to quantitative. Nucleic acids remain in the aqueous phase and proteins separate into the organic phase or lie at the phase interface.

General Procedure

It is typically easiest to carry the extraction out in 1.7–2 mL eppendorf tubes.

Dilute your nucleic acid sample to 100–700 µL or divide your samples into tubes such that you have no more than 700 µL per tube. It is difficult to do the extraction with volumes smaller than 100 µL. The sample can be concentrated again after precipitation.

Add an equal volume of phenol to the tube, vortex vigorously to mix the phases.

Spin in a microfuge at top speed for 1–2 min to separate the phases.

Remove the aqueous phase to a new tube, being careful not to transfer any of the protein at the phase interface.

Repeat the phenol extraction two more times.

Extract the sample two times with an equal volume of chloroform:isoamyl alcohol to remove any trace phenol.

Precipitate the nucleic acid. (nucleic acid precipitation)

Reagents

  • Phenol equilibrated to pH 7.5
  • Chloroform:isoamyl alcohol in a 24:1 ratio (CHIZAM! in Joyce lab lingo)
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