ThisisamodificationofasaltingoutprocedureasdescribedbyMilleretal.,(1988),evaluatedattheDNALaboratory,MedicalSchool,Malta.Whenanalysedbyspectrophotometry>95%ofextractedgenomicDNAgaveapurityratioofDNAtoproteinsintherangeof1.7-1.9andaconcentrationof100ng/µl.

InthefollowingprotocolDNAwasextractedfromperipheralbloodleucocytesusing3mlsofwholeblood.Itwasobservedthatonprolongedstorageofwholebloodat-20and-80ºC,DNAyielddecreasedconsiderablyprobablyduetodegenerationofthewhitebloodcells.Volumesofbuffersandreagentsusedmaybeadjustedaccordingtovolumeofwholebloodused.

PROCEDURE:

1. WholebloodwascollectedindisodiumEDTAcontainersandstoredat-20ºCandanumberofsampleswerealsostoredat-80ºC.TofacilitatehaemolysisofRBCsitisrecommendedtostoreafreshsampleforafewhoursinafreezerasfreezingdestroystheredcells.

2. Afterthawing,3mlsofwholebloodaretransferredtoasterileconicalcentrifugetube(15mlvolume)towhich9mlsof1xerythrocytelysingbuffer(0.155MNH₄Cl;10mMKHCO₃;0.1mMNa₂EDTA;pH7.4)mustbeadded.

3. Thesolutionisleftfor10minutesatRTwithoccasionalmixingbyinversionfollowedbycentrifugationfor5minutesat4000rpm.

4. Aftercentrifugationthesupernatantisdiscardedandawhitepelletwillbeobservedatthebottomofthetube.Thispelletmustbewashedforatleast3timesbyadding3mlsoferythrocytelysingbufferandrepeatingsteps3and4.Itisimportanttobreakdownthepelletandrinseitwellinerythrocytelysingbufferinordertocleanthewhitebloodcellsfromremainingheme.

5. 1.5mlsofSEbuffer(75mMNaCl;25mMNa₂EDTA;pH8.0)containing100mg/mlofProteinaseKand1%sodiumdodecylsulphate(SDSw/v),areaddedtothepellet.Thetubesarethenincubatedatatemperatureof37-55ºC(optimaltempforProteinaseKactivity)overnightinawaterbathorincubator.Duringthisstepthewhitebloodcells"membranesaredenaturedandDNAgoesoutinsolution.

6. Aftertheincubation,1.5mlsofSEbuffertogetherwith750mlof6M(saturated)NaClareaddedtoeachtube,followedbytheadditionof3.75mlschloroform.

7. Thetubesaremixedvigorously(onavortex)forabout20secwithoccasionalmixingforatleast30min.Alternativelyyoucanleavethetubesonarotatorfor1hour.

8. Theemulsionwillthenbecentrifugedfor10minutesat2000rpmwithminimalbreakingforce.Aftercentrifugation2phasesareobservedandcaremustbetakennottodisturbtheinterphase.DuringthisstepDNAisextractedintothesupernatantandproteinsseparatedintothelowerphase.

9. Theupperaqueousphase(containingtheDNA)istransferredintoacleanandsterileconicalcentrifugetubeusingasterilePasteurPipette,followedbytheadditionofanequalvolumeofisopropanol(doublethevolumeof100%ethanolcanalsobeused)

10. DNAwillbeprecipitatedbygentleswirlingofthetubeandisobservedvisuallyasawhitethreadlikestrand.

11. UsingasterilespatulaorlooptransfertheDNAstrandintoasterilemicrocentrifugetubecontaining1mlof75%ethanol.

12. TheDNAisthenwashedbyinversiontocleanitfromanyremainingsaltsandthetubecentrifugedat11000gfor4minutes.Thesupernatantisdiscardedtakingcarenottodiscardthepellet.Repeatthissteponcemore.

13. Afterdiscardingthesupernatantthepelletisdriedfromexcessethanoleitherbyusingavacuumcentrifugeorbyleavingthetubesopenandinvertedinanovenataround50-65ºCforanhour.

14. ThedriedpelletisresUSPendedinTEbuffer(1MTris-HCl;0.5MEDTA;pH8.0)andleftovernightonarotator.

15. DNAconcentrationisdeterminedeitherbyagarosegelelectrophoresisorspectrophotometryandadjustedtothedesiredconcentrationbyaddingmoreTEbuffer.ItisimportanttonotethatbeforeadjustingandreADIngDNAconcentrationsonemustobtainahomogeneoussampleofDNAwhichisnotquiteeasyacquiredsinceDNAisveryviscous.ToadjusttolowerconcentrationonemustusedotherquantitationmethodssuchasPicogreen(Molecularprobes)usingspectrofluorometryasspectrophotometryisnotaccurateandsensitiveatverylowconcentrations.

Fig.1.Col1a1Sp1FragmentamplifiedbyPCRusingDNAextractedbytheabovemethod.

DNASTORAGE²

Forroutinestoragethebestconditionisat+4degreesCelsiusinTEbuffer(pH8.0).HighlypurifiedDNAcanbestoredforlongertime.

DriedDNApelletscanbestoredat-20degreesCelsiusforupto6months.

DNAprecipitatedinethanolcanbestoredindefinitelyat-20degreesCelsius.Forlongtermstorage-80degreesCelsiusisrecommended.

REFERENCE

1.MillerSA,DykesDD,PoleskyHF1988AsimplesaltingoutprocedureforextractingDNAfromhumannucleatedcells.NucleicAcidsRes16:1215.

2.Greenetal.1998GenomeAnalysis:ALaboratoryManual.ColdSpringHarborLaboratoryPress.