PCR产物纯化回收试剂盒操作方法及步骤说明书相关交流-蚂蚁淘在线

PCR产物纯化回收试剂盒操作方法及步骤说明书

蚂蚁淘 /发布时间:1545759609 /浏览量:248

PurificationofPCRProductsinPreparationforCloning

JosephSambrookPeterMaccallumCancerInstituteandTheUniversityofMelbourne,AustraliaDavidW.RussellUniversityofTexasSouthwesternMedicalCenter,DallasExcerptedFromMolecularCloning:ALaboratoryManualThirdEdition

ABSTRACT
TheresidualenzymaticactivityofThermostableDNApolymerasesthatsurvivetherigorsofPCRcancompromisesubsequentenzymaticreactions.ThisprotocoldescribeshowtouseproteinaseKtodestroythermostableenzymesandtopurifyamplifiedDNAinpreparationforcloning.

MATERIALS
ReagentsandSolutions
Ammoniumacetate(10M) AmplifiedDNAfrompolymerasechainreactions Chloroform Ethanol Phenol:chloroform(1:1,v/v) TE(pH8.0)
EnzymesandBuffers ProteinaseK(20mg/ml) GenomicgradeproteinaseKthathasbeenshowntobefreeofDNaseandRNaseactivity.
Gels EthanolAgarosegelcontaining0.5µg/mlethidiumbromide

METHOD
PooluptoeightPCRs(400µl)containing1µgofthedesiredamplificationproduct.(SeeTroubleshooting.)IfmineraloilwasusedtopreventevaporationduringPCR,centrifugethepooledsamplesbrieflyandtransferthelower(aqueous)phasetoafreshmicrocentrifugetube. Add0.2volumeof5xproteinaseKbufferandproteinaseKtoafinalconcentrationof50µg/ml.Incubatethemixturefor60minutesat37°C. InactivatetheproteinaseKbyheatingto75°Cfor20minutes. Extractthereactionmixtureoncewithphenol:chloroformandoncewithchloroform. Add0.2volumeof10Mammoniumacetateand2.5volumesofethanol.Mixthesolutionwellandstoreitfor30minutesat4°C. RecovertheDNAbycentrifugationatmaximumspeedfor5minutesat4°Cinamicrocentrifuge.Discardthesupernatantandthenwashthepelletwith70%ethanol.Centrifugeagain,removethesupernatant,andthenallowtheDNAtodry.TheDNAmaybefurtherpurifiedbychromatographyorbygelelectrophoresis.ThisstepisrecommendedwhenprimershavebeenusedtoaddrestrictionsitestotheendsoftheamplifiedDNA.Unusedprimersandprimer-dimersshouldberemovedbeforedigestingtheDNAwiththeappropriaterestrictionenzymes(pleaseseeRemovalofOligonucleotidesandExcessdNTPsfromAmplifiedDNAbyUltrafiltration). DissolvethepelletinTE(pH8.0).AssumethattherecoveryofamplifiedDNAis50-80%anddissolvetheDNAinTE(pH8.0)atanestimatedconcentrationof25µg/ml(25ng/µl). Analyzeapprox.25ngofthepurifiedDNAbyagarose-ethidiumbromidegelelectrophoresis,usingMarkersofanappropriatesize.Checkthattheamplifiedbandfluoresceswiththeintensityexpectedofapprox.25ngofDNA.
TROUBLESHOOTING
Nonspecificamplificationproductsarepresentatsignificantlevels(e.g.,aredetectablebygelelectrophoresis).Step1Purifythedesiredproductbyelectrophoresisthroughlow-melting-temperatureagarosebeforeproceeding(pleaseseeRecoveryofDNAfromLow-melting-temperatureAgaroseGels:OrganicExtraction).

REFERENCES
CroweJ.S.,CooperH.J.,SmithM.A.,SimsM.J.,ParkerD.,GewertD.1991.Improvedcloningefficiencyofpolymerasechainreaction(PCR)productsafterproteinaseKdigestion.NucleicAcidsRes.19:184-184.[FreeFullText]WybranietzW.A.andLauerU.1998.Distinctcombinationofpurificationmethodsdramaticallyimprovescohesive-endsubcloningofPCRproducts.BioTechniques24:578-580.[Medline]

Anyoneusingtheproceduresinthisprotocoldoessoattheirownrisk.ColdSpringHarborLaboratorymakesnorepresentationsorwarrantieswithrespecttothematerialsetforthinthisprotocolandhasnoliABIlityinconnectionwiththeuseofthesematerials.Materialsusedinthisprotocolmaybeconsideredhazardousandshouldbeusedwithcaution.Forafulllistingofcautionsregardingthesematerial,pleaseconsult:MolecularCloning:ALaboratoryManualThirdEdition,byJosephSambrookandDavidW.Russell,©2001byColdSpringHarborLaboratoryPress,ColdSpringHarbor,NewYork,p..8.25-8.26.

Copyright©2006byColdSpringHarborLaboratoryPress.Allrightsreserved.Nopartofthesepages,eithertextorimagemaybeusedforanypurposeotherthanpersonaluse.Therefore,reproductionmodification,storageinaretrievalsystemorretransmission,inanyformorbyanymeans,electronic,mechanical,orotherwise,forreasonsotherthanpersonaluse,isstrictlyprohibitedwithoutpriorwrittenpermission

================ 蚂蚁淘在线 ================

免责声明：本文仅代表作者个人观点，与本网无关。其创作性以及文中陈述文字和内容未经本站证实，对本文以及其中全部或者部分内容、文字的真实性、完整性、及时性本站不做任何保证或承诺，请读者仅作参考，并请自行核实相关内容