Deproteinationusingphenol/chloroform(Maniatisetal.,1982)

"Phenol"asusedisAnalargrade.Phenolshouldbemeltedat65°C,8-hydroxyquinolineaddedtoafinalconcentrationof0.1%,andequilibratedthreetimeswithanequalvolumeof1MTris.HCl,pH7.0.

ThefinalTriswashisreplacedwithTE(10mMTris,1mMEDTA,pH8.0)andthephenolstoredinthedarkat4°C.8-hydroxyquinolineisaddedtopreventoxidationofthephenolandtoactasaninhibitorofRNases.

"Chloroform"asusedisawatersaturated24:1(v/v)mixtureofchloroformandisoamylalcohol.Thepresenceofisoamylalcoholisrequiredtoaidseparationofthetwophasesduringextractionofaqueoussolutionswithchloroform.Phenol:chloroformisa1:1(v/v)mixtureoftheabovetworeagents.

N.B.:Phenolishighlytoxic.Glovesshouldbewornatalltimeswhenhandlingsolutions/mixturescontainingphenol.Chloroformisahepatic(liver)toxinandalcoholisflammable.

Nucleicacidsarepurifiedasfollows:-

1)Addanequalvolumeofphenol:chloroformtothemixturetobecleanedandvortexedtoemulsifythe2phases.

2)Thephasesareseparatedbycentrifugationat13,000rpmfor2minutesinamicrofugeor5minutesat3,000ginaMSECentaurforlargervolumes.

3)Theaqueousupperphaseisremovedtoacleantube,takingcaretoavoidanyinterfacematerial,andtheextractionrepeateduntilnointerfacematerialisvisIBLe.

4)Theaqueousphaseisthenextractedoncewithanequalvolumeofchloroformandthenucleicacidsalcoholprecipitated.

Ifprecipitationisunnecessarytheaqueousphaseisextractedoncewithanequalvolumeofwatersaturateddiethylether.Theupperetherlayerisdiscardedandanyresidualetherisremovedbyheatingthesampleto65°Cfor5minutes.

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