重组DNA的分离克隆与测序实验手册.docx
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重组DNA的分离克隆与测序实验手册.docx
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重组DNA的分离克隆与测序实验手册
PROTOCOLSFORRECOMBINANTDNAISOLATION,CLONING,andSEQUENCING重组DNA的分离、克隆与测序实验手册
BruceA.Roe,JudyS.Crabtree,AkbarS.Khan
DepartmentofChemistryandBiochemistry,TheUniversityofOklahoma
Norman,Oklahoma73019
Introduction
Thismanualisacompilationofmanyoftheeverydaymethodsusedintheaveragemolecularbiologylaboratory,withemphasisonthetechniquesforlargescaleDNAsequencingprotocolsandDNAsequencingautomationtechniques.Themanualhasbeenwritteninaprotocolformat,withlittletheoreticaldiscussion.Fortheoryandadditionalinformation,usersofthismanualarereferredbacktotheoriginalliterature,ortoothertextualmanualssuchasthosepublishedbyManiatis
(1)etal.andGlover
(2).
Thefollowingpersonsareacknowledgedforcontributingmethodsandsuggestionsduringtheassemblyofthismanual:
StephanieChissoe,SandyClifton,DennisBurian,RickWilson,Din-PowMa,JamesWong,LeslieJohnston-Dow,ElaineMardis,ZhiliWang,KalaIyer,SteveToth,GoughayZhang,HuaQinPanandothermembersoftheRoelaboratory,bothpastandpresent.
1.Sambrook,J.,Fritsch,E.F.,andManiatis,T.,inMolecularCloning:
ALaboratoryManual.ColdSpringHarborLaboratoryPress,NY,Vol.1,2,3(1989).
2.Glover,D.M.DNACloningVolumeI:
APracticalApproach.IRLPress,Oxford,1985.
I.Generalmethods
A.PhenolextractionofDNAsamples
PhenolextractionisacommontechniqueusedtopurifyaDNAsample
(1).Typically,anequalvolumeofTE-saturatedphenolisaddedtoanaqueousDNAsampleinamicrocentrifugetube.Themixtureisvigorouslyvortexed,andthencentrifugedtoenactphaseseparation.Theupper,aqueouslayercarefullyisremovedtoanewtube,avoidingthephenolinterfaceandthenissubjectedtotwoetherextractionstoremoveresidualphenol.Anequalvolumeofwater-saturatedetherisaddedtothetube,themixtureisvortexed,andthetubeiscentrifugedtoallowphaseseparation.Theupper,etherlayerisremovedanddiscarded,includingphenoldropletsattheinterface.Afterthisextractionisrepeated,theDNAisconcentratedbyethanolprecipitation.
Protocol
1.AddanequalvolumeofTE-saturatedphenoltotheDNAsamplecontainedina1.5mlmicrocentrifugetubeandvortexfor15-30seconds.
2.Centrifugethesamplefor5minutesatroomtemperaturetoseparatethephases.
3.Removeabout90%oftheupper,aqueouslayertoacleantube,carefullyavoidingproteinsattheaqueous:
phenolinterface.Atthisstagetheaqueousphasecanbeextractedasecondtimewithanequalvolumeof1:
1TE-saturatedphenol:
chloroform,centrifugedandremovedtoacleantubeasabovebutthisadditionalextractionusuallyisnotnecessaryifcareistakenduringthefirstphenolextraction.
4.Addanequalvolumeofwater-saturatedether,vortexbriefly,andcentrifugefor3minutesatroomtemperature.Removeanddiscardtheupper,etherlayer,takingcaretoremovephenoldropletsattheether:
aqueousinterface.Repeattheetherextraction.
5.EthanolprecipitatetheDNAbyadding2.5-3volumesofethanol-acetate,asdiscussedbelow.
B.ConcentrationofDNAbyethanolprecipitation
Typically,2.5-3volumesofanethanol/acetatesolutionisaddedtotheDNAsampleinamicrocentrifugetube,whichisplacedinanice-waterbathforatleast10minutes.Frequently,thisprecipitationisperformedbyincubationat-20Covernight
(1).TorecovertheprecipitatedDNA,thetubeiscentrifuged,thesupernatantdiscarded,andtheDNApelletisrinsedwithamorediluteethanolsolution.Afterasecondcentrifugation,thesupernatantagainisdiscarded,andtheDNApelletisdriedinaSpeedy-Vac.
Protocol
1.Add2.5-3volumesof95%ethanol/0.12MsodiumacetatetotheDNAsamplecontainedina1.5mlmicrocentrifugetube,inverttomix,andincubateinanice-waterbathforatleast10minutes.Itispossibletoplacethesampleat-20degCovernightatthisstage.
2.Centrifugeat12,000rpminamicrocentrifuge(Fisher)for15minutesat4degC,decantthesupernatant,anddraininvertedonapapertowel.
3.Add80%ethanol(correspondingtoabouttwovolumeoftheoriginalsample),incubateatroomtemperaturefor5-10minutesandcentrifugeagainfor5minutes,anddecantanddrainthetube,asabove.
4.PlacethetubeinaSavantSpeed-VacanddrytheDNApelletforabout5-10minutes,oruntildry.
5.AlwaysdissolvedriedDNAin10mMTris-HCl,pH7.6-8.0,0.1mMEDTA(termed10:
0.1TEbuffer).
6.ItisadvisabletoaliquottheDNApurifiedinlargescaleisolations(i.e.100ugormore)intoseveralsmall(0.5ml)microcentrifugetubesforfrozenstoragebecauserepeatedfreezingandthawingisnotadvisable.
Notesonprecipitationofnucleicacids
A.Generalrules
Mostnucleicacidsmaybeprecipitatedbyadditionofmonovalentcationsandtwotothreevolumesofcold95%ethanol,followedbyincubationat0to-70degC.TheDNAorRNAthenmaybepelletedbycentrifugationat10to13,000xg.for15minutesat4degC.Asubsequentwashwith70%ethanol,followedbybriefcentrifugation,removesresidualsaltandmoisture.
ThegeneralprocedureforprecipitatingDNAandRNAis:
1.Addone-tenthvolumeof3MNaOAc,pH5.2*tothenucleicacidsolutiontobeprecipitated,
2.Addtwovolumesofcold95%ethanol,
3.Placeat-70degCforatleast30minutes,orat-20degCovernight.
oralternatively
1.Combine95mlof100%ethanolwith4mlof3MNaOAc(pH4.8)and1mlofsterilewater.Mixbyinversionandstoreat-20degC.
2.Add2.5volumesofcoldethanol/acetatesolutiontothenucleicacidsolutiontobeprecipitated.
3.Placeatat-70degCforatleast30minutesor-20degCfortwohourstoovernight.
*5MNH4OAc,pH7.4,NaClandLiClmaybeusedasalternativestoNaOAc.DNAalsomaybeprecipitatedbyadditionof0.6volumesofisopropanol.
B.Oligonucleotides
Addone-tenthvolumeof3MNaOAc,pH6.5,andthreevolumesofcold95%ethanol.
Placeat-70degCforatleastonehour.
C.RNA
Addone-tenthvolumeof1MNaOAc,pH4.5,and2.5volumesofcold95%ethanol.
Precipitatelargevolumesat-20degCovernight.
Smallvolumesamplesmaybeprecipitatedbyplacinginpowdereddryiceordryice-ethanolbathforfiveto10minutes.
D.IsobutanolconcentrationofDNA
DNAsamplesmaybeconcentratedbyextractionwithisobutanol.Addslightlymorethanonevolumeofisobutanol,vortexvigorouslyandcentrifugetoseparatethephases.Discardtheisobutanol(upper)phase,andextractoncewithwater-saturateddiethylethertoremoveresidualisobutanol.Thenucleicacidthenmaybeethanolprecipitatedasdescribedabove.
E.Notesonphenolextractionofnucleicacids
Thestandardandpreferredwaytoremoveproteinsfromnucleicacidsolutionsisbyextractionwithneutralizedphenolorphenol/chloroform.Generally,samplesareextractedbyadditionofone-halfvolumeofneutralized(withTEbuffer,pH7.5)phenoltothesample,followedbyvigorousmixingforafewsecondstoformanemulsion.Followingcentrifugationforafewminutes,theaqueous(top)phasecontainingthenucleicacidisrecoveredandtransferredtoacleantube.Residualphenolthenisremovedbyextractionwithanequalvolumeofwater-saturateddiethylether.Followingcentrifugationtoseparatethephases,theether(upper)phaseisdiscardedandthenucleicacidisethanolprecipitatedasdescribedabove.
A1:
1mixtureofphenolandchloroformalsoisusefulfortheremovalofproteinfromnucleicacidsamples.Followingextractionwithphenol/chloroform,thesampleshouldbeextractedoncewithanequalvolumeofchloroform,andethanolprecipitatedasdescribedabove.
C.Restrictiondigestion
Restrictionenzymedigestionsareperformedbyincubatingdouble-strandedDNAmoleculeswithanappropriateamountofrestrictionenzyme,initsrespectivebufferasrecommendedbythesupplier,andattheoptimaltemperatureforthatspecificenzyme.Theoptimalsodiumchlorideconcentrationinthereactionvariesfordifferentenzymes,andasetofthreestandardbufferscontainingthreeconcentrationsofsodiumchloridearepreparedandusedwhennecessary.TypicaldigestionsincludedaunitofenzymepermicrogramofstartingDNA,andoneenzymeunitusually(dependingonthesupplier)isdefinedastheamountofenzymeneededtocompletelydigestonemicrogramofdouble-strandedDNAinonehourattheappropriatetemperature.Thesereactionsusuallyareincubatedfor1-3hours,toinsurecompletedigestion,attheoptimaltemperatureforenzymeactivity,typically37degC.SeetheAppendixforalistingofrestrictionsitespresentintheM13(pUC)MCSandalistingofvariousrestrictionenzymes,incubationconditionsandcutsites.
Protocol
1.Preparethereactionforrestrictiondigestionbyaddingthefollowingreagentsintheorderlistedtoamicrocentrifugetube:
sterileddH20q.s(where"q.s."meansquantitysufficient)
10Xassaybufferone-tenthvolume
DNAxul
restrictionenzyme*yul(1-10unitsperugDNA)
Totalvolumezul
*Ifdesired,morethanoneenzymecanbeincludedinthedigestifbothenzymesareactiveinthesamebufferandthesameincubationtemperature.
Note:
ThevolumeofthereactiondependsontheamountandsizeoftheDNAbeingdigested.LargerDNAsshouldbedigestedinlargertotalvolumes(between50-100ul),asshouldgreateramountsofDNA.
Refertothevendor'scatalogforthechartofenzymeactivityinarangeofsaltconcentrationstochoosetheappropriateassaybuffer(10XHigh,10XMedium,or10XLowSaltBuffers,or10XSmaIBufferforSmaIdigestions).RestrictionenzymesarepurchasedfromBethesdaResearchLaboratories,NewEnglandBiolabs,orUnitedStatesBiochemicals.
2.Gentlymixbypipettingandincubatethereactionattheappropriatet
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