1)ExcisethebandofinterestfromtheTAEgelusingacleanscalpelbladeandplaceinapre-weighedEppendorftube. 2)Add3volumesof6MNaI,0.1%sodiumthiosulphatesolutionandallowagarosetomelt(approx.5minuteswithvortexing).ForTBEgels,0.1volumesof1Mmannitolshouldalsobeaddedtoaidgelsolubilisation. 3)VortexglasssUSPension(finelycrushedglassscintillationvialsuspended1:1insterilenanopureH2OorFlukaanalyticalfilteraidresuspended1:4insterilenanopureH2O)andadd7.5ultoagarosesolution(7.5ulshouldbeusedforupto5ugDNAandthereafteranextra1ulshouldbeusedforeachextraugDNA). 4)AllowDNAtobindfor15-20minutesatroomtemperature. 5)Spindownglass-DNAfor30secs.inamicrofugeatmaximumspeedandcarefullyremovesupernatant.Discardsupernatant. 6)Washpelletin500ul4.5MNaI,0.1%sodiumthiosulphate,re-pelletanddiscardsupernatant. 7)Washpellet2x500ulin1xTE,pH7.5,200mMNaCl,60%EtOH.Afterlastcentrifugation,removealltraceofthesupernatantandallowtoair-dryfor5minutes. 8)EluteDNAat37-55°Cin25ulTE,pH8.0for5minutes. N.B:ItisimportanttoeluteinabufferwithapHofca.8aselutioninwaterorlowerpHbuffersdecreasestheyieldmarkedly. 9)Spindownglassfor5minutesatmaximumspeedinamicrofugeandcarefullyremovesupernatanttoacleaneppendorftube. 10)Repeatelutionstepandpoolsupernatants.Discardpellet.Respinpooledsampletoremovetracesofglassfor5minsandtransfercleansupernatanttoafresh,sterileeppendorftube. Allreagentsaremadefromthehighestgradechemicalsavailable(esp.importantfortheNaI).NaIsolutionsweremadewithsterile,nano-pureH2Oandfinalethanolwasmademinusethanol,autoclavedandethanoladdedtoafinalconcentrationof70%(v/v)aftersterilisation.Glass"beads"weremadefromafinelycrushedscintillationvial(i.ehighqualityglass)bycrushingwithapestleandmortar.Glassiscrushedbasicallyuntilyourwristfeelslikeit"sabouttofalloff...andthensome(shouldbehavelikecookingflour).Alternatively,IhaverecentlytriedCeliteAnalyticalFilterAid(CeliteAFA,Cat.No.22137-Ithinkthatsthecataloguenumberanyhow!)fromFlukainplaceofthecrushedglasswithbrilliantresults.Gene-Kleenprotocol
PurificationofDNAfromagarosegelsisanessentialmethodinvolvedinthesub-cloningofDNAfragments.ThefollowingmethoddescribesavariationofthemethodofVogelsteinandGillespie,1979(Proc.Natl.Acad.Sci.USA.76,(2)615-619).