酚氯仿法提取DNA原理及方法

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说明：本原理及方法是个人整理，用来给研究生教学用的，用本方法可以提取到理想的DNA。各试验室提供的细胞裂解液浓度各有不同，只要经过试验证明的，都可以用来提取到理想的DNA.

1.ExperimentalPrinciples

1)Celllysis(lysisbuffer,containingSDS,EDTA,Tris-HCl,andRNase)

SDS,adetergentisaddedtothebuffertobreakopenthecellmembranes;italsohelpsremoveproteinsandlipidsinthecell.Ethylenediaminetetraaceticacid(EDTA),achelatortoremovemetalionsinsolutiontoprevenDNasefromcuttinguptheDNA.RNaseisalsopresentinthebufferatthisstep,tobreakuptheRNApresentinthecells.

2)Removeprotein

ProteinaseK,itremainsactiveatelevatedtemperatures,sothesolutioncanbeheatedtoabout55°Ctoaidproteininactivationandremovalbythedetergent.3)ExtractDNAfrombuffer

OncethecellsarebrokenopenandtheRNA,proteins,andlipidshavebeendissolvedinthebuffer,theDNAmustbeseparatedfromthesematerials.

Phenol:removetheproteins,leavingDNAandotherwater-solublematerialsbehindbycentrifugation.TheDNAisthenextractedfromthewaterphaseusingchloroformandprecipitatedfromthechloroformusingethylalcoholmixedwithsodiumacetatesalt.4)DNAprecipitation

TheethanolcanprecipitateDNAfromwaterphase

2.MaterialsandSolutions

Allreagentsareprecooledorkeptat4°Cbeforeuse.1)ProteinaseK

2)PhenolsaturatedwithTE(pH8.0)3)Chloroform4)Isoamylalcohol

5)RNasestock(30mg/ml,CatalogNo.R4642-10MG,Sigma)6)10%SDS

7)0.5mol/LEDTA,PH=8.08)1mol/LTris-HCl,PH=8.09)1mol/LNaCl

10)Extractionsolution(ES)(100mMEDTA,200mMNaCI,50mMTris-HCI(pH8.0),0.5%SDS,50μg/mlRNase)

0.5mol/LEDTA,PH=8.01mol/LTris-HCl,PH=8.01mol/LNaCl10%SDSRNasestock(30mg/ml)DDWater1L200ml50ml200ml50ml1.666ml498.3ml50ml10ml2.5ml10ml2.5ml83.3μl25ml3.Experimentalprotocol

1)HarvestcellsandwashcellswithPBS(~106cells)2)Suspendcellsinto500μlES

3)Slowlyadd10μlproteasesK(5mg/ml,Finalconcentrationof100μg/ml)totheabovecellsuspensionwhilegentlymixing.Incubatethissolutionat55°Cforaminimumof2-3hwithoccasionalmanualormechanicalgentlemixing.4)Anequalvolumeofphenol(500μl)isaddedtothecelllysate.Centrifugeat12,000rpmfor5mintoseparatethetwophases.Theaqueous(top)phaseistransferredtoanewtubeusingawideboretransferpipette.

Note:cutatipusingscissorsorbladetogetawideborepipette.

5)AddanequalvolumeofPhenol/chloroform/isoamylalcohol(500μl)intotheaqueousphase,andcentrifugeat12,000rpmfor5mintoseparatethetwophases.Theaqueous(top)phaseistransferredtoanewtube.

6)Addanequalvolumeofchloroform(~500μl)intotheaqueousphase,and

centrifugeat12,000rpmfor5mintoseparatethetwophases.Theaqueous(top)phaseistransferredtoanewtube.

7)Add2volumeofabsoluteethanol(~900μl)totheaqueousphaseandmixgently.Keepat-20°Cfor30min,andcentrifugeat12,000gfor5min.DNApelletshouldbewashedwith70%ethanoltodecreaseresidualsaltandbrieflydriedundervacuumorair-driedat37°Ctoevaporatetheethanol.

8)Theethanol-freeDNAisdissolvedin50μlTE(pH=8.0)orDDwater.

Note:TogethigherconcentrationofDNAsolution,addsmallervolumeTEorwater.

9)MeasuretheabsorbanceofDNAsolutionat260and280nm.ThepuritycanbeestimatedfromtheratioofA260/A280.Aratioof1.8-2.0suggestsminimalproteincontamination.

10)TheDNAsolutionisbeststoredat4°Cor-20°C.

QuickGuideforTraditionalDNAExtractiontechnology

QuickGuideforDNAExtractionKit

Appendix1:Protocolforremovalofparaffin

1)Placeasmallsection(notmorethan25mg)ofparaffin-embeddedtissueina2ml

microcentrifugetube(notprovided).2)Add1200μlxylene.Vortexvigorously.

3)Centrifugeatfullspeedfor5minatroomtemperature.

4)Removesupernatantbypipetting.Donotremoveanyofthepellet.

5)Add1200μlethanol(96–100%)tothepellettoremoveresidualxyleneandmixgentlyby

vortexing.

6)Centrifugeatfullspeedfor5minatroomtemperature(15–25°C).

7)Carefullyremovetheethanolbypipetting.Donotremoveanyofthepellet.8)Repeatstepsstep5–7once.

9)Incubatetheopenmicrocentrifugetubeat37°Cfor10–15minuntiltheethanolhas

evaporated.

10)Resuspendthetissuepelletinlysisbuffer.

Appendix2:Protocolfortissueonglassslides

1)2)3)4)5)

Addadropofabsoluteethanolonslide

Scratchthetissueandtransfertoa2mlmicrotubeEvaporatetheethanolintheairatroomtemperatureAddlysisbufferinto