Reagents

BufferA(Redbloodcelllysisbuffer)composition

• 0.32Msucrose
• 10mMTrisHCl
• 5mMMgCl₂
• 0.75%Triton-X-100

AdjustpHto7.6

BufferB(ProteinaseKbuffer)composition

• 20mMTris-HCl
• 4mMNa₂EDTA
• 100mMNaCl

AdjustpHto7.4

N.B.Allsolutionsshouldbesterile.BufferAshouldbeautoclavedpriortoadditionofTriton-X-100.Sterilefilteringofsolutionsinsteadofautoclavingisabetteroption.Procedure

• Add1volumeofbufferAto1volumeofbloodand2volumesofcold,sterile,distilled,deionisedwater.Vortexgentlyorinverttube6-8timesandleavetoincubateonicefor2-3minutes.
• Spinat3500rpmfor15minutesat4^oC.Discardsupernatantinto2.5%bleachsolutionandre-sUSPendpellet(vortexfor30secondsatmediumspeed)in2mlofbufferAand6mlofwater.Spinat3500rpmfor15minutesat4^oC.Thepelletshouldbewhitetocreamincolour.Ifpelletissignificantlyred,repeatwashingstepagain.
• Add5mlofBufferBand500µlof10%SDStopellet.Re-suspendpelletbyvortexingvigorouslyfor30-60seconds.Thenadd50µlofProteinaseKsolution(20mg/ml).TheProteinaseKsolutionshouldbemadefreshandrefrigeratedpriortouse.
• Leavetoincubatefortwohoursat55^oCinawaterbath.Removesamplesandleavetocooltoroomtemperature(orleavefor2-3minutesonice).Add4mlof5.3MNaClsolution.Vortexgentlyfor15seconds.
• Spinat4500rpmfor15-20minutesat4^oC.Pouroffsupernatantintoafreshtube.Takecarenottodislodgepellet.Addanequalvolumeofcoldisopropanol(storedat-20^oC).Invert5-6timesgentlytoprecipitateDNA.
• RemoveDNAwithawideboretipandtransfertoamicrofugetube.Washwith1mlof70%ethanol.LeaveDNAtodryfor15-20minutesat37^oC.Re-suspendin300-400µlofTrisHCl,pH8.5(notTE!).Leavetore-dissolveovernightatroomtemperature.DNAcanbesafelyrefrigeratedforuptoayear.Long-termstoragemayinvolveethanolat-70^oC.

References

• Helms,C.SaltingoutProcedureforHumanDNAextraction.InTheDonis-KellerLab-LabManualHomepage[online].24April1990.[cited19November2002;11:09EST].Availablefrom:http://hdklab.wustl.edu/lab_manual/dna/dna2.html.
• Epplen,J.E.,andT.Lubjuhn.1999.DNAprofilingandDNAfingerprinting.BirhkhauserVerlag,Berlin.p.55.