1.KillcellsatmidlogphasebyaddingNaAzideto0.1%.Immediatelychillculturebyaddingittofrozen0.2MNaEDTA.(40mlofEDTA/200mlofculture)Shakeasyouwouldforamartinitochillthecells.Spin6K5mintopelletcells.

2.Washcellsinice-coldwater.Transfercellstoaconical-bottom50mlFalcon2098tube.(Atthispointyoumayfreezethecellsasa"dry"pelletat-20.)

3.ResUSPendthecellsat1.5to2X10⁹/mlinice-coldNuclearIsolationBuffer(NIB=17%glycerol,50mMMOPSbuffer,150mMpotassiumacetate,2mMmagnesiumchloride,0.5mMspermidine,and0.15mMspermine;pHisadjustedto7.2afterallingredientsaredissolved).

4.Mixcellsuspensionwithanequalvolumeofacidwashedglassbeads(0.45to0.52mmdiameter).Keeponice.

5.Vortexatmaximumspeedonahealthyvortexerfor30secperiods.Chilloniceforatleast30secbetweenepisodesofvortexing.Repeatthevortexing10to15timesoruntilmorethan90%ofthecellshavebeenbroken(monitorbycheckingforthefractionofghostsinthephasecontrastscope).Breakageismostefficientwhentheentirecontentsofthetubeareliftedbytheswirlingaction.Ialsofindthaterringonthesideofhavingtoomanybeadsaidsinbreakage.

6.RemovethesupernatantwithaglasspasteurPipetteorabluepipetemantip--stickthetipthroughthebeadstothebottomofthetubeandsuckuptheliquid.Itdoesn"tmatterifafewbeadsgettransferred.Rinsethebeadstwicewith1.5volumesoffreshNIB.Poolandspinat8KinanSS-34rotorfor20min.

7.Resuspendpelletofnuclei,ghosts,andunbrokencellsatafinalconcentrationof2X10⁹cellequivalents/mlin50mMTris,50mMEDTA,100mMNaCl,pH8.

8.AddSarkosyltoachieveafinalconcentrationof1.5%.Mixgently.

9.AddsolidProteinaseKtoafinalconcentrationof300microgram/ml.Incubateat37^ofor1hr.

10.Centrifuge(cold)at5Kfor5mintopelletcellsandghosts.Thesupernatantshouldhaveonlyminorturbidity.

11.Foreachmlofsupernatantsoution,add1.05gofCsCl.(Iaimforasupernatantvolumeof4.0ml(or4.08g)plus4.2gofCsCl.ThisvolumewillfilloneVTi65heatsealtube.)EncouragetheCsCltodissolvebygentlemixing.Don"tmixvigorouslyorfoamingwillresult.

12.AftertheCsClhasdissolved,measurethevolumeandadd0.025volumesofa5mg/mlstocksolutioninwaterofHoechst33258dye.Mix.

13.Transferto5mlheat-sealtubes.Fillthetubetothetopwithadditional"dummy"solution.("dummy"isthesamesolution--NaCl,Tris,EDTA,CsCl,Sarkosyl,Hoechst--minustheyeastDNA).

14.SpininaVTi65orVTi65.2rotorat55Kand20^ofor18-24hr.

15.VisualizetheDNAbandsunderlongwaveUVlight.Theoreticallytherearethreebands.Thetop,diffusebandismitochondrialDNA.TheprominantbandischromosomalDNAandthefaintbandimmediatelybelowistherDNAband.(HoechstseparatesDNAbasedonGCcontent.)Youmaynoticeabandofparticulatejunkbetweenthenuclearandmitochondrialbands.Stayawayfromthisstuff.RemovetheDNAbysidepunctureusinga16gaugeneedle.Measurethevolumeinthesyringebeforedispensingitintoa15mlcorextube.(Iusallyremovetheneedlebeforedispensingittoreducethepossibilityofaddedshear.)

16.Addanequalvolumeof5:1isopropanol:H₂Osolution.Swirlthetubetomixthecontents(Idon"tusethevortexerbecauseofworryaboutshear).Afterthephasesseparate,removethealcoholphasewithapasteurpipette.Iletthephasessettleoutinthedrawnouttipofthepipetteanduseitlikeaseparatoryfunneltominimizethelossofanyaqueousphase.Repeattheisopropanolsteptwicemore.

17.Add3volumesofcold70%ethanolslowly,tothesideofthetube.WiththeethanolfloatingontheCsCllayerbeginmixingthephaseswithasinglequickswirlingmotion.TheDNAshouldbegintocomeoutofsolutionattheinterfaceasafibrousnetwork.ContinuethesingleswirlsuntilthephasesaremixedandtheDNAhasfallenoutofsolution.TheDNAcanberemovedbytouchingtheDNAclotwiththeendofapasteurpipettethathasbeenclosedbymeltingitinaflame.TheDNAwillsticktotheglassandcanbesubmergedinfresh70%ethanoltorinsetheclotandthenteasedfromtheendoftheglasstipontothesideofamicrofugetube.Don"tlettheclotdryoutcompletelybeforeaddingTEtoredisolvetheDNA.Ifyouarelookingatplasmidreplicationintermediates,spintheethanol-CsClsolution20minat8K.Rinsethetubewithfresh70%ethanol,airdrythetubeandresuspendanyDNAinTE.ThetwoDNAsolutionscanthenbepooled.Fora500mlcultureIwouldresuspendtheDNAinafinalofabout400microliters.Itmaytaketheclotseveraldaystogointosolution.IfyouareworriedaboutlosingsmallbubblesyoumightwanttoaddNaClto50mMaftertheDNAhasbegungoingintosolution.

18.TolookatsinglecopysequencesIusuallycutabout1microgramofthenuclearDNAin0.12to0.15mlofbufferwith4-5microlitersofenzymefor5hours.DigestionmaybecompletewithlessenzymeorwithshorterincubationsbutsinceitislikelytovarywiththeenzymeIhaven"tstrayedmuchfromthisplan.TheDNAisprecipitatedbytheadditionof2volumesofabsoluteethanolcontaining0.5MK-acetate,chillingfor15minat-70^o,andspinning15mininamicrofuge.Afterrinsingthepelletin70%ethanolanddryingthetubeIresuspendthepelletdirectlyinloADIngbuffer.