Genomic DNA prep
来自 : 蚂蚁淘
GenomicDNAprep(JorgensenLab[modifiedslightlybyMikeH.])- Makewormgrowthplates.Theseuseagarosetoavoidimpuritiesinmostbatchesofagar,andareenrichedtoallowgreaterwormgrowth.
Mix:5gBactotryptone2gBactoyeastextract5gNaCl20gagarose1LdH2O
Autoclavethissolution.
Add:1ml5mg/mlcholesterolinethanol2ml1MCaCl21ml1MMgSO425ml1MKH2PO4
Pourthemediaintolargeplates(about20mlperplate),andletdry.
SpreadtheplateswithHB101,oranotherhighgrowthstrainofE.coli,e.g.AMA1004
- Addwormstotheplates,andletthemgrowuntiltheplatesstarve.
- RinsethewormsoffinM9,putthemin15mlpolypropylenetubes,andpelletthembyspinning30sec.inaclinicalcentrifuge.
- Removethesupernatent,andwashandpelletthewormsinabout4mlofTEN.TEN:20mMTris50mMEDTA100mMNaCl,pH7.5
- Removethesupernatent,andresUSPendthewormsinabout0.5mlTEN.Freezeat-20°C.Frozenwormsseemtobeeasiertodigest.(Thewormscannowbestoredat-20°indefinitely,sothisisagoodplacetosynchronizeDNAprepsofdifferentstrainsofworms.)
- TransferthewormsinTENtoa1.5mlEppendorftube.Add:25
l10%SDS(_>0.5%SDS)2.5l20mg/mlProteinaseK(_>0.1mg/ml) - Incubatethetubesat50°Cto60°Cforonehour.Resuspendthepelletat10min.,20min.,and30min.
- Addanother2.5lofProteinaseKandincubateforanotherhour.
- Phenolextractthesolutionswith0.5mlphenol.Mixwellbyinversion.
- Phenol/CIAextractthesolutions.(CIA=24:1Chloroform:isoamylalcohol)
- CIAextractthesolutions.
- Add45lof3MNaOAc,and0.8mlethanolatroomtemperature.
TheDNAshouldprecipitatealmostimmediately.Mixthetubesbyinversion,andflickthemwithyourfingertowindtheDNAstrands.
- Spinverylightly(2-5sec.)inamicrofugetoprecipitatetheDNA.
(Longerspinsmaybringdownotherstuffaswell.)
- Draintheethanol,andwashthepelletinicecold70%ethanol.
- Drainthewashandletthetubesdrycompletely.
Aspirateoffanydropsofethanolwithapulled-outpasteurPipette.
- Resuspendthepelletsin0.5mlTEN.
(Thisisoftenadifficultresuspension.Heatingto50°Cmayhelp.Timealsohelps,sothisisagoodplacetostopfortheday,andputyourtubesat4°C.)
- Add2lof10mg/mlRNaseA(_>40mg/ml).
- Incubateat37°Cforonehour.
- Phenolextractthesolutions.
- Phenol/CIAextractthesolutions.
- CIAextractthesolutions.
- Add45lof3MNaOAc,and0.8mlethanolatroomtemperature.
TheDNAshouldprecipitatealmostimmediately.Mixthetubesbyinversion,andflickthemwithyourfingertowindtheDNAstrands.
Youmaywanttoleavethetubesat-20°for30min.toovenightatthispoint.
- Spinthetubes30sec.inamicrofuge.
- Draintheethanol,andwashthepelletwithicecold70%ethanol.
- Drainthewashandletthepelletdrycompletely.Aspirateoffanyliquid.Resuspendthepelletin100lofTE(10,1).
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