CTABTECHNIQUE/Method/Schedule/Protocol(JPB)FORDNAISOLATION/DNAEXTRACTIONFROMPLANTLEAF/LEAVESSAMPLES

(seealsoDNARNAdoubleisolationprocedureifbothDNAandRNAareneeded)

Reagentsneeded

CTABbuffer

2%CTAB20gmCTAB

20mMEDTA40mlEDTAstock(0.5M)

100mMTris-ClpH8.0100mlTris-Clstock(1M)

1.4MNaCl280mlNaClstock(5M)

makeupto1Litrewithwater,pH7.5-8.0,andautoclave

+0.2%Mercaptoethanol

WashBuffer

76%Ethanol

10mMNH4Ac

DNAExtraction

1.Preheat5mlCTAB(add10µlmercaptoethanoltoeach5mlCTAB)inablue-topped50mlcentrifugetubeat60-65oC.Removeanddiscardmidribs,andwraplaminaeinaluminiumfoilandfreezeinliquidnitrogen.0.5–1.0gmtissue/5mlCTAB

(CanstoreleafmaterialafterliquidNitrogen–1-2daysat–20or–80forlongerperiods)

2.Gentlycrumbleleaftissueovercoldpestleofliquidnitrogen.Grindfrozenleafwithonespatulaoffinesandadd0.5spatulaofPVPPpowderaftergrinding.

3.Scrapepowderintodrytubeandaddpre-heatedbufferandmixgently.Avoidleavingdrymaterialaroundrimoftube.AdjustCTABvolumetogiveaslurry-likeconsistency,mixoccasionally.

4.Incubatefor60minat60oC

5.Addequalvolumeofchloroform/iso-amylalcohol(24:1),Mixforabout3min,thentransfercontentstonarrowborecentrifugetubes.Balancebyaddingextrachlor/iso.Spin5,000rpmfor10min(ensurecorrecttubesused),brakeoff.(ForextrapureDNAisolation-spinandretainsupernatentbeforechloroformextraction).

6.Removesupernatantwithwide-borepastette(cutoffbluetip)tocleantube,repeatchloroformextractiononce.Supernatentshouldbeclear,thoughmaybecoloured.

7.PrecipitateDNAwith0.66vol.ofcoldisopropanol-canleaveovernight.SpooloutorspindownDNA,2minat2,000rpm.

8.Transferto5mlwashbufferfor20min.

9.DrybrieflyandresUSPendin1mlT.E.(canbeleftovernight)

10.Add1µl10mg/mlRNAsetoeach1mlT.E./DNAmixtureandincubatefor60minat37oC.(IfRNASeinthesampledoesn’tmatter–stages11and12maybeomitted)

11.Dilutewith2volumesT.E.andadd0.3vol3MSodiumacetate

(pH8)+2.5volcold100%ethanol,

12.SpoolDNAout.Airdryandresuspendinin0.5to1mlT.E.(takestime)andfreezeuntilrequired.

DNAQuantification

Makea0.8%agarosegelwith1xTAEand0.1µlofEthidiumbromideper10mlsolution).Loadsamplesneatandata1in10(1+9)dilution.,with3µlloADIngbuffer.AlsoincludeaLamdaladdercutwithHindIIIandEcoRI.Thiscontains100ngofDNApermicrolitreanduseasfollows:

1µlladder+4µlwater+2µlloadingbuffer

2µlladder+3µlwater+2µlloadingbuffer

ThedifferentbandsoftheladderareofknownmolecularweightandknownDNAconcentration.Matchthebrightnessofyoursampleswiththoseofthetwodilutionsoftheladder.Refertothediagramtomatchthebandwiththeconcentration.Rememberthatalthoughtheladderconcentrationsareabsolute,youhaveloaded5µlofsampleandalsodilutedsomeofthem.Thismustbetakenintoaccountwhencalculatingthestrengthofthesamplesinng/µl.

Pestlesandmortarswashedfor20-30minin0.25MHCl,rinsedinwaterandair-dried,allmesstobetidiedupandtubeswashedandlefttodrain.