1.Growcellsatleast7generationsin

0.1%(w/v)[¹³C]-acetateorglucose

0.01%(w/v)[¹⁵N]-ammoniumsulfate

Aminoacids/supplements(asrequired,addedtothesameconcentrationasinnormalsyntheticmedium)

--dissolvedin"-N"mediumandfiltered.(Iadd0.1mg/mlampicillinifI"mfeelingparanoid.)

ThedoublingtimeofRM14-3ais~2.6hrat23^oCinglucoseand6-8hr(dependingonaeration)inacetate--longerifyou"reselectingforaplasmid.RM14-3aisMATa,cdc7-1^ts,bar1,trp1-289,leu2-3,112,ura3-52,his6inanA364Abackground.

Iaimforabout32x10⁶cellspertimepoint.ThatgivesenoughDNAforatleast3blots,leavingenoughleewayforerrors.

2.Whenthecelldensityis2x10⁶cells/ml(OD660~0.16),arrestwith200nMalphafactor(1-1.25doublingtimes,until%unbuddedcellsstopsincreasing).NOTE:Thisalphafactorconcentrationisforbar1strains.BAR1+strainsrequire15xmorealphafactor.

3.Filter,washinYcomp+alphafactor,resUSPendinYcomp+[¹²C]-glucose+alphafactor.

[Idon"tparticularlytrytoresuspendthecellsinexactlythesamevolumetheywereinoriginally,butrather,inavolumethat"sconvenient.]

4.Transferto37^oC,waitforculturetoreach37^oC,thenaddPronase(0.01-0.05mg/mlfinalconc.).[YoucanadddryPronase.IusuallydissolvethePronasein5mlofminimalmedium,justtoavoidhavingthedryPronasesticktothesidesoftheflask.]

5.Holdat37^oCfor90-120min(untilbuddedcellsaccumulate).Removeonesample(t=0),thenswirltheflaskinice-waterandreturnitto23^oC.Continuetocollectsamples(untilt=~80min).

Theice-watertreatmentistoquick-chillthecultureto23^oC,andisusuallyfor~1min.Youcanempiricallydeterminehowlongittakestobringyourdesiredvolumeofmediumfrom37^oto23^oC.

6.Collectingsamples:Freeze8mlof0.1%Na-azide,0.2MEDTAin"50ml"Oakridge-typeplastic,screw-cappedcentrifugetubes(frozenslantedtomaximizethesurfacearea).Duringtheexperiment,mix20mlofcellswith1/50volumeof10%Na-azideina35mlCorextube(onice)andimmediatelytransferthemixtoatubeoffrozenEDTA/azide.Alternatively,squirtthe10%azideonthefrozenazide/EDTAandimmediatelyaddthecellsample.VortexorshakethetubevigorouslytochillthesampleandbreakupthefrozenEDTA.Spindownthecellsinachilledcentrifuge,washwith1mlcoldwaterpersampleinEppendorftubesandfreezethepelletsat-20^oC.

7.ExtractDNA(smash-and-grabwithglassbeads),digestwiththeenzymeofchoicein0.1mlofreactionmixpersample.[Iresuspendthenucleicacidspelletin0.05mlof10mMTris,0.1mMEDTA,andaddanequalvolumeofa2xenzymemixthatcontainstheappropriaterestrictionenzymebuffer,restrictionenzyme(usually1microliterpersample)and0.25microgram/mlRNaseA.IftheDNAprepisclean,thedigestcangoovernightat37degreesC.]

Choosingarestrictionenzyme:thefragmenttobeprobedshouldideallybe3-15kb,toavoidbroad,overlappingheavy-heavyandheavy-lightDNApeaksintheCsClgrADIent.

8.Checkforcompletionofthedigestbyrunning8-10microlitersofitonagel.Tobecertain,thegelshouldbeblottedandprobedforaknownfragment.However,ifyouarefamiliarwiththepatternexpectedforaparticularrestrictionenzyme(e.g.,EcoRI),youmaybeabletodecidejustfromtheethidiumbromide-stainedgelwhetherthedigestwascomplete.Igenerallyblotandprobethegelanyway.

9.Mixeachsample(90-92microliters,restrictionenzymeandall)with9.141gofCsClsolution,togivearefractiveindexof1.4042-1.4043.(Remember,it"saloteasiertodiluteasolutionthat"stoodensethantomakeatoo-dilutesolutionmoredense!)

CsClsolution:

Use1.28gdryCsClpergramof10mMTris,100mMEDTA,pH7.5.TheT₁₀E₁₀₀shouldhavearefractiveindexof1.3395.Itmaytoohighwhenyoufirstmakeit(upto1.4010orthereabouts);justadd10mMTrisuntiltherefractiveindexcomesdown.AftertheCsClisinsolution,therefractiveindexshouldbe1.4052.It"sworthadding90microlitersofdummyrestrictionenzymemixto9.141goftheCsClsolutionjusttomakesurethatyougettherightrefractiveindex.ThisdummyCsClmixthenservestotopofftherealsamples.

Note:Calibratetoarefractiveindexof1.3330fordistilledwater.9.141gCsClsolution=5.25ml.IfindthatIgetmuchmoreconsistentreadingsbyweighingouttheCsClsolution(insmall,sterileculturetubes)thanbypipettingitout.Iinitiallyused5.4mlCsCl(=9.402g;DNAscaledtomatch),butthatleavesabout0.25-0.3mlsolutionleftoveraftertheQuicksealtubesarefilled.With5.25mlCsCl+0.1mlDNA,youmayfindthatthereisafair-sizedbubbleatthetopoftheQuicksealtube.Thisisokay--usethedummymixtobringupthevolumeandbalancethetubes.

10.Spinat50000rpmfor18hr,28000rpmfor3.5hr,nobrake(i.e.,deceleration=0)ateitherstep(usedualprogram).

11.Fractionatethegradients(10dropsperfraction,collectedinmulti-wellmicrotitreplates),readtherefractiveindexofevery3rdor4thfractionassoonaspossIBLethereafter.

12.HHDNApeaksatrefractiveindexofabout1.405,andHLat1.4040,soyoushouldusefractionsfrom~1.4060-1.4030.Igofor24fractions(totakeupthe24slotsperlaneoftheslotblot),soyoumayhavetojuggleabittogettherangeyouwant.Alternatively,youcouldloadallthefractionsandavoidthisproblem,butthentheslotblotbecomesmorecomplex.

Use42microlitersofeachfractiontobeblotted,andmicrotitreplatesforthedenaturation/neutralizing:

Mix42microlitersofDNA(inCsCl)with28microlitersof1NNaOH(=>0.4NNaOHfinalconc.).Sealtheplatewithanadhesivesheetandincubateat65^oCfor1hr.

Cooltoroomtemperatureandneutralizewithanequalvolume(70microliters)of20xSCP.Pre-wetthemembranein10xSCP.Run140microlitersof10xSCPthroughtheslotsandthenapplythesample.Runanother140microlitersof10xSCPthrough--togetthelastbitofthesampleoutofthemicrotitrewell,Ifirstaddthis10xSCPtothewellwherethesampleusedtobe,thenapplyittotheslotblot.RemovethemembraneandUVcrosslinkimmediately.Iletcross-linkedmembranessitin6xSCP,1%Sarkosyl,0.1%BSA(dilutedfromCalbiochem"s30%solution)whilethenextonesarebeingdone.

Note:IfindthatIcansaveonyellowtipsbyusingthesameset(onamultichannelpipettor)severaltimes,rinsingthetipsoutbypipettingupanddownwithwater(inaPyrexdish)betweensamples.(I"vedonethetestofrinsingandthenusingthesametipstoapply10xSCPtoalaneoftheslotblot;Igotnohybridizationthere,indicatingthattherewasnocarry-overofsample.Youmaychoosetousefreshtips--therewasonenightwhenIhadthelastboxoftipsandwasforcedtoimprovise!)

13.Pre-hybridizeandhybridizeasusual.

WeroutinelyusetwoorthreesequencesasourMarkersforearlyandlatereplication.Previously,weusedtocutplasmidDNAtoobtainfragmentstolabelasprobes.Thesedays,weuseasetofPCRprimersandyeastgenomicDNAtoamplifythefragmentsofinterest.OurstandardmarkersequencesandtheirPCRprimersare:

ARS305(chromosomeIII,veryearly-replicating,mayshowsome"escape"replicationatthecdc7block;seeReynoldsetal.,1989,Mol.Cell.Biol.9:4488).

Primers:

5"-ATTCGCCTTCTGACAGGACG-3"

5"-ATAACGGAGACTGGCGAACC-3"

GAL3(chromosomeIV,ARS1-adjacent,early-replicating;seeMcCarrollandFangman,1988,Cell54:505).

Primers:

5"-CCTACATGGCCATAGATCCG-3"

5"-AAGGTGGATAGTGCAACCGC-3"

R11(chromosomeV,late-replicating;seeFergusonetal.,1991,Cell65:507).

Primers:

5"-GATTGCACGACCAACTTCGG-3"

5"-GTTAGGTAGAGCAGGCAGAC-3"

ThequantitationmethoddescribedbelowisbasedonAppleMacintoshapplicationsandfileformats;comparableoptionsmaybeavailableontheWindowsplatformaswell.

14.Thehybridizationontheslotblotisquantitatedbysomemeans(weuseaPackardInstantImageroraMolecularDynamicsPhosphorImager),andthehybridizationintensitiessoobtainedareplottedforeachtimepoint(i.e.,eachgradient).

Typicalgradientprofileslooksomethinglikethis(inthisexample,theslotblotswereprobedwithaRAP1fragmentandquantitatedonaPhosphorImager):

15.TheplotforeachtimepointissavedasaPICTfile(ifyouuseKaleidaGraph,asetofplotscanbesavedasonePICTfile).

16.TheHHandHLpeaksarequantitatedbymeasuringtheareaundereachpeak.ThePICTfilesareopenedinNIHImage,andtheareaundereachpeakismeasuredbytracingthepeakwiththefreehandtoolandapplyingthe"Measure"command.BecausetheHHandHLpeakstendtooverlap,youwillhavetoexercisesomejudgementintracingtheoverlappingportionsofthecureves.Weusetheouterhalfofeachpeakasaguideandtraceasymmetricallinedowntheoverlappingportion.Anexampleofthecurveswewouldtypicallytrace:

17.Theextentofreplicationateachtimepointiscalculatedfromtheequation