QIAquick_Gel_Extraction_Kit_Protocol

ProtocolQIAquickSpinHandbook03/200123QIAquickGelExtractionKitProtocolusingamicrocentrifugeThisprotocolisdesignedtoextractandpurifyDNAof70bpto10kbfromstandardorlow-meltagarosegelsinTAEorTBEbuffer.Upto400mgagarosecanbeprocessedperspincolumn.ThiskitcanalsobeusedforDNAcleanupfromenzymaticreactions(seepage8).ForDNAcleanupfromenzymaticreactionsusingthisprotocol,add3volumesofBufferQGand1volumeofisopropanoltothereaction,mix,andproceedwithstep6oftheprotocol.Alternatively,usethenewMinEluteReactionCleanupKit.Notes:•TheyellowcolorofBufferQGindicatesapH≤7.5.•Addethanol(96–100%)toBufferPEbeforeuse(seebottlelabelforvolume).•Isopropanol(100%)andaheatingblockorwaterbathat50°Carerequired.•Allcentrifugationstepsarecarriedoutat≥10,000xg(~13,000rpm)inaconventionaltable-topmicrocentrifuge.•3Msodiumacetate,pH5.0,maybenecessary.1.ExcisetheDNAfragmentfromtheagarosegelwithaclean,sharpscalpel.Minimizethesizeofthegelslicebyremovingextraagarose.2.Weighthegelsliceinacolorlesstube.Add3volumesofBufferQGto1volumeofgel(100mg~100µl).Forexample,add300µlofBufferQGtoeach100mgofgel.For2%agarosegels,add6volumesofBufferQG.ThemaximumamountofgelsliceperQIAquickcolumnis400mg;forgelslices400mgusemorethanoneQIAquickcolumn.3.Incubateat50°Cfor10min(oruntilthegelslicehascompletelydissolved).Tohelpdissolvegel,mixbyvortexingthetubeevery2–3minduringtheincubation.IMPORTANT:Solubilizeagarosecompletely.For2%gels,increaseincubationtime.4.Afterthegelslicehasdissolvedcompletely,checkthatthecolorofthemixtureisyellow(similartoBufferQGwithoutdissolvedagarose).Ifthecolorofthemixtureisorangeorviolet,add10µlof3Msodiumacetate,pH5.0,andmix.Thecolorofthemixturewillturntoyellow.TheadsorptionofDNAtotheQIAquickmembraneisefficientonlyatpH≤7.5.BufferQGcontainsapHindicatorwhichisyellowatpH≤7.5andorangeorvioletathigherpH,allowingeasydeterminationoftheoptimalpHforDNAbinding.5.Add1gelvolumeofisopropanoltothesampleandmix.Forexample,iftheagarosegelsliceis100mg,add100µlisopropanol.ThisstepincreasestheyieldofDNAfragments500bpand4kb.ForDNAfragmentsbetween500bpand4kb,additionofisopropanolhasnoeffectonyield.Donotcentrifugethesampleatthisstage.ProtocolQIAquickSpinHandbook03/2001246.PlaceaQIAquickspincolumninaprovided2mlcollectiontube.7.TobindDNA,applythesampletotheQIAquickcolumn,andcentrifugefor1min.Themaximumvolumeofthecolumnreservoiris800µl.Forsamplevolumesofmorethan800µl,simplyloadandspinagain.8.Discardflow-throughandplaceQIAquickcolumnbackinthesamecollectiontube.Collectiontubesarere-usedtoreduceplasticwaste.9.(Optional):Add0.5mlofBufferQGtoQIAquickcolumnandcentrifugefor1min.Thisstepwillremovealltracesofagarose.ItisonlyrequiredwhentheDNAwillsubsequentlybeusedfordirectsequencing,invitrotranscriptionormicroinjection.10.Towash,add0.75mlofBufferPEtoQIAquickcolumnandcentrifugefor1min.Note:IftheDNAwillbeusedforsaltsensitiveapplications,suchasblunt-endligationanddirectsequencing,letthecolumnstand2–5minafteradditionofBufferPE,beforecentrifuging.11.Discardtheflow-throughandcentrifugetheQIAquickcolumnforanadditional1minat≥10,000xg(~13,000rpm).IMPORTANT:ResidualethanolfromBufferPEwillnotbecompletelyremovedunlesstheflow-throughisdiscardedbeforethisadditionalcentrifugation.12.PlaceQIAquickcolumnintoaclean1.5mlmicrocentrifugetube.13.ToeluteDNA,add50µlofBufferEB(10mMTris·Cl,pH8.5)orH2OtothecenteroftheQIAquickmembraneandcentrifugethecolumnfor1minatmaximumspeed.Alter-natively,forincreasedDNAconcentration,add30µlelutionbuffertothecenteroftheQIAquickmembrane,letthecolumnstandfor1min,andthencentrifugefor1min.IMPORTANT:EnsurethattheelutionbufferisdispenseddirectlyontotheQIAquickmembraneforcompleteelutionofboundDNA.Theaverageeluatevolumeis48µlfrom50µlelutionbuffervolume,and28µlfrom30µl.ElutionefficiencyisdependentonpH.ThemaximumelutionefficiencyisachievedbetweenpH7.0and8.5.Whenusingwater,makesurethatthepHvalueiswithinthisrange,andstoreDNAat–20°CasDNAmaydegradeintheabsenceofabufferingagent.ThepurifiedDNAcanalsobeelutedinTE(10mMTris·Cl,1mMEDTA,pH8.0),buttheEDTAmayinhibitsubsequentenzymaticreactions.