Gene-Kleenprotocol

PurificationofDNAfromagarosegelsisanessentialmethodinvolvedinthesub-cloningofDNAfragments.ThefollowingmethoddescribesavariationofthemethodofVogelsteinandGillespie,1979(Proc.Natl.Acad.Sci.USA.76,(2)615-619).

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.

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