免疫共沉淀(CoIP)_图文
来自 : 蚂蚁淘
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| Author:Laura-LeeBoodram |
| Source:Laura-LeeBoodram,DepartmentofLifeSciences,TheUniversityoftheWestIndies |
| Abstract:Theprotocolissimpleandfairlyrapid.Itdoesnotrequiretheuseoforganicsolventsbutratherutilizessaltextractiontoprecipitatecontaminatingproteins.HighqualityDNAisobtainedsuitableforimmediatePCRapplications.Onecanobtainapproximately100-200ugofDNAfrom4-8mLoffreshorfrozenwholeblood. |
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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.
- Spinat3500rpmfor15minutesat4oC.Discardsupernatantinto2.5%bleachsolutionandre-sUSPendpellet(vortexfor30secondsatmediumspeed)in2mlofbufferAand6mlofwater.Spinat3500rpmfor15minutesat4oC.Thepelletshouldbewhitetocreamincolour.Ifpelletissignificantlyred,repeatwashingstepagain.
- Add5mlofBufferBand500µlof10%SDStopellet.Re-suspendpelletbyvortexingvigorouslyfor30-60seconds.Thenadd50µlofProteinaseKsolution(20mg/ml).TheProteinaseKsolutionshouldbemadefreshandrefrigeratedpriortouse.
- Leavetoincubatefortwohoursat55oCinawaterbath.Removesamplesandleavetocooltoroomtemperature(orleavefor2-3minutesonice).Add4mlof5.3MNaClsolution.Vortexgentlyfor15seconds.
- Spinat4500rpmfor15-20minutesat4oC.Pouroffsupernatantintoafreshtube.Takecarenottodislodgepellet.Addanequalvolumeofcoldisopropanol(storedat-20oC).Invert5-6timesgentlytoprecipitateDNA.
- RemoveDNAwithawideboretipandtransfertoamicrofugetube.Washwith1mlof70%ethanol.LeaveDNAtodryfor15-20minutesat37oC.Re-suspendin300-400µlofTrisHCl,pH8.5(notTE!).Leavetore-dissolveovernightatroomtemperature.DNAcanbesafelyrefrigeratedforuptoayear.Long-termstoragemayinvolveethanolat-70oC.
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.
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