mTn-lacZ/LEU2

Uses:Genedisruption,analysisofgeneexpression,immunodetectionofbeta-galfusionprotein.

Inmoredetail:mTn-lacZ/leu2wasconstructedbySiefertetal.(1986).Itcaneasilybeinsertedatmutiplesitesinagivengene.ThemutagenizedDNAisthentransformedintoyeast,whereitreplacesthechromosomallocusbyhomologousrecombination.Thetransposoninsertionscreateapoolofinsertion/disruptionalleles.Insertionsthatgeneratein-framefusionofthecodingregiontolacZcanbeusedtomonitorandquantifygeneexpression,viaassaysforbeta-galactivity.Thefusionproteincanalsobeimmunodetectedusingantibodiesdirectedagainstbeta-gal.

TheaccessionformTn-lacZ/LEU2isU35112

AkitformutagenesisofayeastgenewithmTn-lacZ/LEU2isavailable.

• Seewhatitcontains
• Orderthekit

Pleasereadthiswholedocumentbeforeyoustart!

Shuttlemutagenesis

1. ClonefragmentintovectorpHSS6(fromstrainR1123;mapgivenbelow).
   • DeleteasmuchofthepolylinkeraspossIBLeassometimestransposon"hot-spots"intoit.SelecttransformantsonLBKan40.
2. TransformthisplasmidintocompetentcellsofR1236/B211.
   • SelectonLBKan40Cm34.
3. TransferF::mTn-lacZ/LEU2intocellsbymatingwithstrainB198
   • Growstrainsovernightwithantibioticselection(Amp50forB198).
   • Subculture1:100infreshmedium(noantibiotics).Growat37^oCtoearlylogphase(whencellswirlsarevisible).Therecipientstrain(B211)canbedenserthanthedonor.
   • Mix200ulofeachstrain.Incubateat37^oCwithoutagitationfor20minto1hr.Plateas100ulaliquotsontoLBAmp50Kan40Cm34.
   • Grow1-2daysat30^oC.Nowhavecointegrates.(SetupstrainR1230inSm50overnight).
4. MatetostrainR1230toresolvecointegrates
   • Elutecoloniesfromplates:put2mlsofLBontheplate,scrapeoffthecolonieswithaspeader.Thisisyoureluate.Youshouldhaveseveralthousandcoloniesatleast.
   • DiluteovernightcultureofstrainR12301:100withoutantibiotic.Diluteeluatetoroughlysamedensity.
   • Growandmateasbefore.
   • Aftermatingfor20minto1hr,plate100ulaliquotsonLBAmp50Kan40Sm50andgrowovernightat37^oC.
   • DotheControl:Spotthestartingstrainsontothismedia.
5. RescueresolvedDNAfromthisstrain
   • EluteyourcoloniesoffinLB.Again,youshouldhavethousands.DilutesomeeluateinLBAmp50Kan40togiveanalmostsaturateddensity.Growat37^oCforafewhours.
   • IsolateDNAbyminiprep.(Wedoastandard1-2-3alkalinelysisbutuse150ulof7.5MNH4AcassolutionIII,and270ulofisopropanoltoprecipitate.Thisremovesmostofprotein(avoidingphenol)andRNA,givingaverysmallcleanpellet.Still,therearenucleasessowekeepeverythingonice).
   • Transformabout1/10ofminprepintoaregularrecAendAcloningstrain(egDH5).PlateonLBAmp50Kan40.
6. TransformintoyeastselectingforLEU2
   • Eluteentirepooloftransformants(again,aimforthousands)andmakeaminiprepasinstep5.(Make-70stockofbacterialpoolforfutureuse).
   • TransformNotIdigestofentirepoolintoyeastandlookforlacZfusionsamongtransformants.

Screeningforin-framelacZfusionsinyeast

1. TransformantcoloniesarepatchedtoSC-leu.
2. CellsarereplicaplatedtoaSC-leuplateandaSC-leuplateonwhichasterilediscofWhatman1Afilterpaperhasbeenplaced,andgrownovernightat30^oC.Othermediaorgrowthconditionscanbesubstitutedasdesired.Forade2strains,anytestmediashouldcontain80mg/lofadenine,astheredpigmentcanobscuretheX-galresult.
3. Filtersareliftedfromtheplatesandplacedinthelidofa9-cmglasspetridish.Thislidisthenplacedinsideaclosed15-cmglasspetridishcontainingchloroform,for10to30minutes.Theminimumexposuretimenecessaryforaparticularyeaststraincanbedeterminedempirically.
4. Filtersareplacedcolony-sideupontoX-Galplates(120ug/ml5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside,0.1MNaPO4[pH7]and1mMMgSO4in1.6%agar)andincubatedat30^oCforupto2days.
5. TransformantscarryingproductivelacZfusionsarerecoveredfromtheregrownSC-leuplate.ItisadvisabletosubsequentlymaintainselectionforLEU2whereverpossible,assomemutationsaredeleteriousevenintheheterozygousstate.

Bacterialstrainsused(includedinkit):

Vectorused:

TheaccessionforpHSS6isM84115

Antibioticsused:

Whenonlyafewplatesofeachtypeareused,it"sconvenienttochopanLBplateupwithasteriletoothpick,putthebitsinasterileflask,andmelttheagarbymicrowave.Addappropriateamountsofantibioticandrepourplates.

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