GenomicDNAprep(JorgensenLab[modifiedslightlybyMikeH.])

1. Makewormgrowthplates.Theseuseagarosetoavoidimpuritiesinmostbatchesofagar,andareenrichedtoallowgreaterwormgrowth.

   Mix:5gBactotryptone2gBactoyeastextract5gNaCl20gagarose1LdH₂O

   Autoclavethissolution.

   Add:1ml5mg/mlcholesterolinethanol2ml1MCaCl₂1ml1MMgSO₄25ml1MKH₂PO₄

   Pourthemediaintolargeplates(about20mlperplate),andletdry.

   SpreadtheplateswithHB101,oranotherhighgrowthstrainofE.coli,e.g.AMA1004

2. Addwormstotheplates,andletthemgrowuntiltheplatesstarve.
3. RinsethewormsoffinM9,putthemin15mlpolypropylenetubes,andpelletthembyspinning30sec.inaclinicalcentrifuge.
4. Removethesupernatent,andwashandpelletthewormsinabout4mlofTEN.TEN:20mMTris50mMEDTA100mMNaCl,pH7.5

5. Removethesupernatent,andresUSPendthewormsinabout0.5mlTEN.Freezeat-20°C.Frozenwormsseemtobeeasiertodigest.(Thewormscannowbestoredat-20°indefinitely,sothisisagoodplacetosynchronizeDNAprepsofdifferentstrainsofworms.)
6. TransferthewormsinTENtoa1.5mlEppendorftube.Add:25l10%SDS(_>0.5%SDS)2.5l20mg/mlProteinaseK(_>0.1mg/ml)

7. Incubatethetubesat50°Cto60°Cforonehour.Resuspendthepelletat10min.,20min.,and30min.
8. Addanother2.5lofProteinaseKandincubateforanotherhour.
9. Phenolextractthesolutionswith0.5mlphenol.Mixwellbyinversion.
10. Phenol/CIAextractthesolutions.(CIA=24:1Chloroform:isoamylalcohol)
11. CIAextractthesolutions.
12. Add45lof3MNaOAc,and0.8mlethanolatroomtemperature.

    TheDNAshouldprecipitatealmostimmediately.Mixthetubesbyinversion,andflickthemwithyourfingertowindtheDNAstrands.

13. Spinverylightly(2-5sec.)inamicrofugetoprecipitatetheDNA.

    (Longerspinsmaybringdownotherstuffaswell.)

14. Draintheethanol,andwashthepelletinicecold70%ethanol.
15. Drainthewashandletthetubesdrycompletely.

    Aspirateoffanydropsofethanolwithapulled-outpasteurPipette.

16. Resuspendthepelletsin0.5mlTEN.

    (Thisisoftenadifficultresuspension.Heatingto50°Cmayhelp.Timealsohelps,sothisisagoodplacetostopfortheday,andputyourtubesat4°C.)

17. Add2lof10mg/mlRNaseA(_>40mg/ml).
18. Incubateat37°Cforonehour.
19. Phenolextractthesolutions.
20. Phenol/CIAextractthesolutions.
21. CIAextractthesolutions.
22. Add45lof3MNaOAc,and0.8mlethanolatroomtemperature.

    TheDNAshouldprecipitatealmostimmediately.Mixthetubesbyinversion,andflickthemwithyourfingertowindtheDNAstrands.

    Youmaywanttoleavethetubesat-20°for30min.toovenightatthispoint.

23. Spinthetubes30sec.inamicrofuge.
24. Draintheethanol,andwashthepelletwithicecold70%ethanol.
25. Drainthewashandletthepelletdrycompletely.Aspirateoffanyliquid.Resuspendthepelletin100lofTE(10,1).

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