Theoverallsequenceofeventsis:

•Titerandplateoutphage•Liftplaquesontofiltersandpreparethemforscreening•Makeaprobe•Hybridizetheprobetothefilters•Washthefiltersandexposetofilm•Purifyputativeplaques•Exciseplasmidfromthedesiredphage

Somepreparatoryitems

Thelibrary(onethatImadefromC.albicansgenomicDNA)iskeptat-80degreesCin0.5mlaliquots.Thissolutionisalsostableintherefrigeratorforseveralmonthsatleast.Thelibrarymaybeamplifiedifyouwish,butthisshouldnotbenecessary.ItwasmadebycuttingthegenomicDNAwithTsp509Iatseveraldifferentconcentrationsandsize-selectingpiecesfrom4to8-9kbfromeachdigestiontoligateintotheLamBDaZapIIvector(atEcoRI).Itscomplexityisroughly80,000Xaninsertsizeof4to8kb=320Mbp,whichisabout20-foldcoverageofthisyeastgenome.

Theaimistoevenlyplateoutabout10,000phageoverabout10plates.Whatyouneedinadditiontothephageis:Plates:LB,LBtet,LBKan,LBamp;Topagar:NZYorTBplus0.7percentagarautoclave);Cells:XLBlueMRstrain,whichistetracyclineresistant(onanFprimeelement;25micrograms/mltet),andtheSolRstrain,whichiskanamycinresistant(use25microgramspermlaswell).Bothofthesestrainsshouldberecoveredfromthefreezerontotheappropriatedrugplateandthenkeptintherefrigerator.GrowthebacteriainLB+0.2percentmaltose(toexpressthemalpermeasewhichisthelambdareceptor)overnightat30degrees,spindownina50mlFalcontube,andresUSPendtohalfvolumein10mMMgSO4.Thesebacteriawillbegoodtouseforphageplatingforseveralweeks.Use50microlitersperplate.Filters:IlikesimpleunchargednylonfiltersfromFisher(MSIN00HY08250,82mm,50/box).Thesearequitecheap.Solutions:LB,SMsolution,Churchhybridizationsolution,Churchwashsolution,Chloroform(seetheendforrecipes).Ex-assistphageaswellforplasmidexcision(fromStratagene).

Phageplatingunit

• •PutouttherequiredLBplatesto37or30degreesonetothreehoursbeforetheyareneeded.•Whenusingalibraryforthefirsttime,thefirststepistotiterthelibrary,confirmingthetiterstatedbywhoevergaveittoyou.DilutephageinSMsolutiontoaseriesoftestdilutions.•Mixthephagedesired(inabout1to50microliters)with50microlitersofbacteriaandincubateat30degreesor37degreesfor20minutes.•Placeenoughculturetubesat45-48degrees.Thiscaneitherbeinanelectricblockbathwithappropriatelysizedholesorinanad-hocwaterbathoftapwaterinanicebucket.•Meltenoughtopagarinthemicrowavetoaliquotthreemlsintoeachculturetube.•Addthephage/cellstothetopagar,vortexgentlywithoutfrothing,andpourgentlyovertheLBplate,tippingtocoveritcompletely.•Placeat37degrees.Plaqueswillbeapparentby6hoursorsoandreadytoliftafewhoursafterthat.Fortheoriginallibraryplating,youwanttheplaquestobeprettysmall(1to2mm),andyouwillprobablyuseahighenoughdensitysothattheplaqueswillbeconfluent.Forplaquepurification,ontheotherhand,youwillwanttohaverelativelylarge(3mm),well-separatedplaques,whichcanbelefttogoovernightifyouwish.

Plaqueliftunit

• •Putphageplatesto4degreestofirmupthetopagar.•Preparethesolutions;NaOH,Tris,andSSC,andpourthemoutonWhatmanpaper(twolayers)forwettingthefilters.Youwantthepapertobeverywet,butnotwithfreefluidswimmingontop.Whenputtingfilterson,youdon"twantsignificantairbubblesbeneaththem.•Writeanidentifierwithpenciloneachfilter.Considermakingduplicatefiltersforeachplatetopre-verifythatanysignalintheendisreal.•Carefullylaythefilterface-downovertheplaques,thenstabthesandwichwithahigh-guagesyringesoakedinindiainkinfourplacesdowntotheplatebottom.Thiswillprovidegoodalignmentmarksforpullingplaquesintheend.•Carefullypullthefilterbackofftheplate,andlayface-upontheNaOHsolutionpaperfor5minutes(allthesetimesarerough).ThentransfertotheTrisneutralizationpaperfor5minutes,repeatonfreshTrisneutralizationpaper,thento2XSSCfor5minutes.Youmayrepeaton2XSSC.•UV-crosslinkinaStratalinkerwhilethefilterismoist,thensubmergein2XSSC(ormoredilute)toremovedebrisfromthefiltersurface.•Storedryforthelong-term,orwetforimmediateuse.

Hybridizationunit

• •Forfreshfilters,prehybeinChurchplusBSAfor30minutesormore,warm.Oldfiltersdonothavetobeprehybe"d,butmayneedtobeboiledinthepresenceofthenewprobetoremovetheoldprobe.•Sealinarotorunit,orifyouhavealotoffilters,youneedtouseaseal-a-mealbag,withabout1.5to2mlhybesolutionpersmallplate-sizedfilter.(Iuse20mlfor12filters)•Putto65degreesfor3hourstoovernightorlonger,withsufficientagitationtoexposeallthefilters.•Savethehybridizationsolutionona50mlFalcontubeforfutureuse.Washthefiltersthreetimesfiveminuteswithchurchwashatroomtemperatureinaseparatecontainer.ThefirstwashisdisposedinrADIoactivewaste,subsequentonesgodownthesink(withrinsing).•LayoutonafilmbackandexposetoX-OmatARfilmfortimeasindicatedbythegeigercounter.•Makesuretoattachphosphorescentorotheralignmentmarkstoyourassemblytoallowprecisealignmentintheend.

Plaquepullingunit

• •Congratuations!Yougotsignal.Nowyouneedtopulltheplaqueorplaqueareatorecoverandpurifythephage.Gotothelightboxwiththeautoradiogram,thefilterassembly,andamarkingpen.Alignthefilterassemblyoverthefilmandmarkpositivesignalsonthetopsaranwrap.Nowgetouttheoriginalplatesfromtherefrigeratorandgotoyourdesk.Witharightangleprotractor,aligntheplatewiththefilterassemblybytheirinkmarks,thenmarkthesignallocationonthebackoftheplatewithapen.•PrepareEppendorftubeswith.8mlofSMforeachpostivesignalyouwishtopull.•TakealongpasteurPipetteandsqeezinggentlyonthebulb,jabintomarkedspotontheplate.Nowwithdrawgently,releasingthebulbtoapplyabitofsuction.BlowtheplugintoitsdestinedSMtube.Mixthoroughly/letsitforawhiletoreleasethephage.Thissolutionwillbestableintherefrigeratorforyears,especiallywithadropoffreshchloroformaddedtokillthebacteria.•Nowtopurifythephage,someoftheplugsupernatantsolutionneedstobedilutedandplatedouttoprovidenicelyseparatedplaquesthatcanbere-probed(withthesamehybesolution).Dilute10microliterssupernatantto1mlofSM,andthenplateout1,5and25microlitersfromthisdilution.Itisbadformtoletphagegrowfortoolong(itleadstomutagenesis).Forlong-termstorageofphage,combinesomephagesuperwith20percentDMSO,andfreeze.Nowgobacktotheotherprotocols.

Plasmidexcisionunit

• •ThebiggestvirtueofthisLambdaZapIIphagelibraryisthatitsinsertscanbeeasilyexcisedintoplasmid(Bluescript)form.Thisprocedureappearsmuchmorecomplicatedthanitactuallyis.FirstyoumusthavetheSolRcellsgrownupandpreparedforphageplating,andgetoutsomeExassistphageaswellfromthefreezer.•Ina15mlfalcontube,combine200microlitersXLbluecellsand200microlitersofphagesupernatantand1microliterExassisthelperphage.Putto37degreesfor15min.•Add3mlLBandputto37degreesfor3hours,shaking(1hourislessefficient,butOK).•Placeto65-70degreesfor20minutestokilloffthebacteria,thenspin15minintheRC3centrifugeattopspeed.•Add100,10and1microliterssupernatantto200microlitersSolRcellsin1.5mltube.Putto37degreesfor15minutes.•PlateonLBampplates.

SOLUTIONS

• •Topagar:Tb-topisasimplerecipe:5gTryptone,2.5gNaCl,and4gagarin500ml-autoclave,andaddMgSO4to10mMandpourinto100mlbottlesforfuturemicrowaving.(Topagarandotherlarge-scalephagegrowthsolutionsdonothavemaltosebecauseitisnotneededwheninfectingcellswithavastlocalexcessofphage,andyoudon"twanttolosephageintheendtobindingtothecelldebris.)•SM:ThisisastABIlizationandstoragesolutionforphage.2.9gNaCl(50mM),1gMgSO4(10mM),25mlTrispH7.5(50mM),50mggelatin(100g/l)in500ml--autoclave.•NaOHforplaquelifts:0.5MNaOH,1.5MNaCl,1mMEDTA.•Trisforplaquelifts:0.5MTrisHClpH8,1.5MNaCl,1mMEDTA.•SSCforplaquelifts:2X,madefrom20Xlabstock-SeeManiatis,etc.•Churchhybridizationsolution:(PNAS81:1992)1percentBSA,1mMEDTA,7percentSDS,and
• 0.5MNaPO4(Froma1.0molarstock,pH7.2:134gNa2HPO4/7H2Oplus4.5mlof85percentphosphoricacidtooneliter;pHcarefully,orelseyourhybesolutionwillbeviscous,andthatiswrong).•Churchwash:50to100mMNaPO4,2percentSDS,1mMEDTA.

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