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Replication timing by density transfer
M.K.Raghuraman 1.Growcellsatleast7generationsin --dissolvedin"-N"mediumandfiltered.(Iadd0.1mg/mlampicillinifI"mfeelingparanoid.) ThedoublingtimeofRM14-3ais~2.6hrat23oCinglucoseand6-8hr(dependingonaeration)inacetate--longerifyou"reselectingforaplasmid.RM14-3aisMATa,cdc7-1ts,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.Transferto37oC,waitforculturetoreach37oC,thenaddPronase(0.01-0.05mg/mlfinalconc.).[YoucanadddryPronase.IusuallydissolvethePronasein5mlofminimalmedium,justtoavoidhavingthedryPronasesticktothesidesoftheflask.] 5.Holdat37oCfor90-120min(untilbuddedcellsaccumulate).Removeonesample(t=0),thenswirltheflaskinice-waterandreturnitto23oC.Continuetocollectsamples(untilt=~80min). Theice-watertreatmentistoquick-chillthecultureto23oC,andisusuallyfor~1min.Youcanempiricallydeterminehowlongittakestobringyourdesiredvolumeofmediumfrom37oto23oC. 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-20oC. 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.).Sealtheplatewithanadhesivesheetandincubateat65oCfor1hr. 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). GAL3(chromosomeIV,ARS1-adjacent,early-replicating;seeMcCarrollandFangman,1988,Cell54:505). R11(chromosomeV,late-replicating;seeFergusonetal.,1991,Cell65:507). 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 Denseisotopes: "-N"medium=1.61gyeastnitrogenbasew/oaminoacidsandammoniumsulfate,11.1gsuccinicacid,6.67gNaOHperlitre. Pronase:WeuseCalbiochem"sPronase(catalog#53702),whichseemsprettyclean--i.e.,platesspreadafterPronasetreatmentaren"tcontaminated--butIheardfromsomeonewho"dusedSigma"sPronaseandhadmassivegrowthofStreptomyces. 20xSCP=0.6MNa2HPO4,2.0MNaCl,pH6.8 T10E100 Addto350mldH2O: Stiruntilthesolidshavedissolved,bringthepHto7.5withconcentratedNaOH(<0.5ml),andbringthevolumeupto500mlwithdH2O.Filter-sterilize;donotautoclave.Therefractiveindexofthesolutionshouldbe1.3393-1.3395. CsCl:fromGallard-SchlesingerIndustries,CarlePlace,NY11514-1731.It"s99.9%pure"SpecialBiochemicalGrade",Catalog#612501,$150/kilogram.
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