请使用支持JavaScript的浏览器! mTn3xHA/lacZ 实验方法_蚂蚁淘,【正品极速】生物医学科研用品轻松购|ebiomall -蚂蚁淘商城
当前位置: > 首页 > 技术文章 >
mTn3xHA/lacZ 实验方法
来自 : 蚂蚁淘

MakinglibraryDNAfromtheDNAwesendyou

Theoriginallibrarywasdistributedas18individualpools.After8/98,thereisanewlibrarythatisdistributedas10pools.YouwillbesentaboutamicrogramofeachpoolintheformofDNA.TransformasuitableamountintoE.coli(useanykanamycin-andtetracycline-sensitivestrainsuitableformakingplasmidpreps).Selecttransformantswith40ug/mlkanamycinand/or3ug/mltetracycline(allowatleastanhourforexpressionfollowingtransformation).Obtain50,000coloniesforeachpool.ElutecoloniesfromplatesinLB;makea-70oCstockofthiseluate.DiluteeluateintoLBplusantibiotictogiveaculturewithanalmostsaturateddensity.Growat37oCforafewhours.MakeminiprepormidiprepDNA.

TransformingyeastwithDNAfromtheinsertionlibrary

OVERVIEW:MutagenizedDNAfromthelibraryisexcisedfromthebacterialvector.Itisthentransformedintoaura3strainofyeast.Thisprocedureisoutlinedinthisfigure.Thebeststrategyistoscreenafewthousandtransformantsfromeachpool.Useofacircle-zerostrainwillpreventrecoveryofinsertionsinthe2-micronplasmid.Forlibrariesdistributedafter8/98,thegenomiclibrarywasmadefromacircle-zerostrain(alsorho-minus)sothisisnotnecessary.Screening30,000transformantsshouldgiveyou95%coverageoftheyeastgenome.

Tominimizedoubleintegrants,transformationsshouldcontainthelowestamountofDNApracticable.Wethereforerecommendthatapilotexperimentbeperformedtodeterminetransformationefficiencyofthestrain,andconditionsthenbescaledupasappropriate.ThepilotprotocolgivenbelowusesamodifiedversionofthemethodofChenetal.(1992).Youshouldusewhatevertransformationprotocolworksbestinyourhands.

  1. PlasmidDNAfrompoolsofthemTn-3xHA/lacZ-mutagenizedgenomiclibraryisdigestedwithNotI.A2.1-kbbandfromthevectorshouldbeveryapparent,togetherwithabroadbandinthe8-kbregion,representinginserts.BecausesizedgenomicDNAwasusedtomakethelibrary,theinsertbandsarenotveryheterogeneousinsize.
  2. A10-mlcultureoftheyeasthoststrainisgrowntoadensityof107cells/ml(O.D.600of1).Useofsuchlogarithmically-dividingculturesincreasestransformationefficiency.
  3. Cellsarepelletedandwashedoncewith5volumesofOneStepbuffer(0.2MLiAc,40%PEG4000,100mMbeta-mercaptoethanol).Thiswashisespeciallyimportantwhenculturevolumesareincreased.
  4. CellsareresUSPendedin1mlofOneStepbuffercontaining1mgofdenaturedsalmonspermDNA.100ulaliquotsofthissuspensionarethenaddedtotubescontainingfrom0.1to1ugofNotIdigestedplasmidDNA.
  5. Tubesarevortexedtomixthecontentsthoroughly,thenincubatedat45oCfor30minutes.
  6. Cellsarepelletedandresuspendedin400ulofSC-ura.200ulisplatedontoSC-uramedium.Platesareincubatedat30oCfor3to4days.

ScreeningforgeneexpressionusinglacZfusions

OVERVIEW:Transformantstrainscarryingin-framefusionsbetweenyeastgenesandlacZareidentifiedbyacolorassayforbeta-galactosidaseactivity.

  1. TomaximizedetectionoflacZfusionsexpressedatalowlevel,transformantcoloniescanbepatchedtoYPADplatesatadensityof100perplate.
  2. Toidentifyvegetativelyexpressedgenes,cellsarereplicaplatedtoanSC-uraplateonwhichasterilediscofWhatman1Afilterpaperhasbeenplaced,andgrownovernightat30oC.Othermediaorgrowthconditionscanbesubstitutedasdesired.Forade2strains,anytestmediashouldcontain80mg/lofadenine.
  3. Filtersareliftedfromtheplatesandplacedinthelidofa9-cmglasspetridish.Thislidisthenplacedinsideaclosed15-cmglasspetridishcontainingchloroformfor10to30minutes.Theminimumexposuretimenecessaryforaparticularyeaststraincanbedeterminedempirically.
  4. Filtersareplacedcolony-sideupontoX-Galplates(120ug/ml5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside,0.1MNaPO4[pH7]and1mMMgSO4in1.6%agar)andincubatedat30oCforupto2days.Theseplatescanbeverythin;theiruseincreasesthesignaloverthatobtainedbysimplysoakingthefiltersinabufferedX-galsolution.
  5. TransformantscarryingproductivelacZfusionsarerecoveredfromtheregrownYPADplates.ItisadvisabletosubsequentlymaintainselectionforURA3whereverpossIBLe,assomemutationsaredeleteriousevenintheheterozygousstate.

Identificationofthegenomicsiteoftransposoninsertion

OVERVIEW:Todeterminethesiteoftransposoninsertion,genomicDNAimediatelyadjacenttothelacZsequencesisrescuedinEscherischiacoli(seefigure).Tointroduceanoriginofreplication(ori),aplasmidmarkedwithLEU2(pRSQ2-LEU2,U64693)replacespartofthetransposonbyrecombinationbetweenplasmid-andtransposon-bornecopiesoflacZsequences.YeastDNAisrecoveredfromthesetransformantsandcutwitha"recovery"enzyme(EcoRI,HindIII,ClaI,SalI,XhoI,KpnI).Thisreleasesasalinearsegmentthebacterialoriginofreplication,thebeta-lactamasegeneandaportionofthelacZgenewithadjacentyeastDNA;thisfragmentisthencircularizedandrecoveredinbacteria.pRSQ2ishighcopynumberinE.coli.Plasmidsaresequencedusingaprimercomplementarytothe5"endofthetransposon.Thisprocessrequiresthethreeprotocolsgivenbelow.

Alternativemethod:CarlFriddle(http://genome-www.stanford.edu/group/botlab)hasdevelopedavectorettePCRrescueprotocolforlacZ-basedtransposons.IhavetranscribedCarl"sprotocolandmodifiedthesuggestedenzymesandprimers,tomakeitsuitableforvectorettePCRofthemTn-3xHA-basedtransposons.

CAUTION!Twoormoreinsertioneventsmayhaveoccurred.Thesecanbeidentifiedbyexaminationofsegregationofthetransposon-borneURA3Markerupontetraddissection.YoushouldbesurethatthestrainhasonlyonetransposonbeforeproceedingtorecoveringaplasmidcontaininggenomicDNA.Ifyouhaveusedthelibraryformutagenesis,youarestronglyadvisedtomakesurethatyourphenotypeislinkedtothetransposoninsertion,sincespontaneousmutationscanariseatothersites.Youcanwastetimerecoveringjunkifyoudon"tcheck.

Transformationofyeaststrains

Wesuggesttransformingtheyeastwith1-5ugofBamHI-digestedpRSQ2-LEUplasmidDNA,selectingthetransformantsonSC-leu-ura.Thisisatargettedreplacement,soefficiencywilldependonyourstrain.ThemethodofChenetal.(1992)givenabovecanbeused.IfanampRplasmidispresentintheyeaststraintobetransformed,adifferentmarkercouldbeclonedintothepRSQ2polylinkertoenableitsrecovery.

RecoveryofgenomicDNAfromyeaststrains

ForadetaileddiscussionofgenomicDNApreparationfromyeast,seePhilippsenetal.(1991).Hereisthemethodthatweuse.

  1. Yeaststrainsaregrowntosaturationat30oCin2mlofYPAD.Wesuggestusingacoupleoftransformantsforeachstrain.
  2. Cellsarerecoveredbycentrifugationat13,000r.p.m.for1minute.Thesupernatantisremovedbyaspirationandcellsareresupendedin250ulof0.1MEDTA(pH7.5),14mM[[beta]]-mercaptoethanolcontaining150ug/mlzymolyase.Cellsareincubatedat37oCuntilspheroplasted.Overspheroplastingdoesnotaffectrecovery.
  3. 50ulofminiprepmix(0.25MEDTA(pH8.5),0.5MTrisbase,2.5%SDS)isaddedtoeachtube.Samplesaremixedbyinversion,thenincubatedinawaterbathat65oCfor30minutes.
  4. 63ulof5MKAcisaddedtoeachsample.Samplesaremixedbyinversionandincubatedonicefor30minutes.
  5. Samplesarespunat13000r.p.m.inamicrofugefor10minutes.Supernatantsaretransferredbypouringeachsampleintoanewtubecontaining720ulof100%ethanol.ADNAprecipitateshouldbevisible.Samplesaremixedbyinversionandspunfor5minutesasabove.
  6. Tubesaredrainedthoroughly,and130ulTEcontaining1mg/mlRNAaseAisaddedtotheundriedpellets.ResupensionofDNAisgradualandoccursduringsubsequentincubationat37oCfor35minuteswithoccasionalvortexing,DNAisreprecipitatedbyadditionof130ulofisopropanol.Samplesaremixedbyinversionandspunfor5minutesasabove.
  7. Tubesaredrained.A70%ethanolwashmaybeperformedtoremovesalt.Finally,pelletsareairdriedandresupendedin40ulofTEwithincubationat37oC.About10ugofgenomicDNAisobtained.

Plasmidrescue

  1. 5ugofyeastgenomicDNAisdigestedovernightat37oCwith5unitsof"recovery"enzyme(e.g.EcoRI,HindIII,SalI,ClaI,XhoI,orKpnI)inavolumeof40ul.
  2. 20ulofthesampleisrunonageltocheckdigestion.Theremainderisheatedto65oCfor25minutestoinactivatetherestrictionenzyme,and215ulofH2O,25ulof10Xligasebufferand1ulofligase(400units)areadded.Tofavourintramolecularreactions,theDNAconcentrationintheligationshouldnotbeover10ug/ml,andcanbeaslowas2ug/ml.
  3. Afterligationat16oCfor4to16hours,DNAisprecipitatedbyadditionof125ulof7.5MNH4Acand375ulofisopropanolandrecoveredbymini-centrifugationat13,000rpmfor20minutes.
  4. TheDNApelletiswashedoncewith70%ethanol,thenresuspendedin6-20ulofTE.3ulofthisistransformedintoE.coli,(weuseelectroporation)selectingforampicillinresistance.Dominiprepsofseveralcoloniesforeachstrain.
  5. Rescuedplasmidscanbeanalyzedbydouble-digestionwithBamHIandthe"recovery"enzyme.Desiredplasmidsdisplaya2.85-kbbandcontainingvectorsequences(3.9kbforEcoRI;seeFigure2)plusadditionalband(s)fromgenomicDNA.Ifyouget"mystery"plasmids,tryadifferenttransformant/recoveryenzyme.
  6. DNApreparationsmaybesequencedusingaprimercomplementarytosequencesjustinsidetheinvertedrepeat(cttctaccttcAATggccgc).Onlytrustyoursequenceuptothefirstsitefortherecoveryenzyme,asotherfragmentscangetclonedinduringcircularization.

SequenceoflacZendofmTn3-3xHA

Uppercaseindicatesterminalrepeatelementalsopresentinvector:

GGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGgcggccattgaaggtagaag

TheaccessionformTn-3xHA/lacZisU54828.

Transferringthedisruptionalleletootherstrains:

WhenpRSQ2-LEU2integratesintomTn-3xHAitcreatesan11.8kbinsertion.Thiselementisnotcleavedbythefollowingenzymes:AscI,AvrII,BspEI,MscI,NotI,PmeI,PmlI,SacII,SnABI,SpeI.Theseenzymescanthereforebeusedtorecoveralargeplasmidcontainingsequencesboth5"and3"tothetransposoninsertion.Wehavesuccessfully"moved"disruptionsbythisstrategy.

UsingtheHATepitopetaggingfeatureofmTn-3xHA/lacZ

Whentransposoninsertionhascreatedanin-framefusiontolacZinthegeneofinterest,thetransposoncanbeexcizedtoleavea274bpinsertion(sequencegivenbelow)containingthe3xHAtag.Withthe5basepairduplicationcausedbytransposoninsertion,thisgivesanin-frame93-aminoacidinsertionintheprotein.ThepopouteventismediatedbycrerecombinaseandrequiresinductionoftheGAL1-10promoterongalactose.Ourstrainsgrowpoorlyongalactosebutgive80to100%popouts.

TheHAtripletagcanbedetectedbymousemonoclonalantibodies12CA5(Boehringer)orMMS101R(BAbCo,Richmond,California).Theseantibodyrecognisecross-reactingyeastproteinsofabout55kDor110kD,respectively,andcangiveaspottybackgroundonimmunofluorescence.Despitethisdrawback,the3xHAtaghasbeenusedextensivelyandsuccessfullyinyeast.Arabbitpolyclonalantiseraisalsoavailable(101c500;BabCo)butthiswaslessreactiveintheoneinstancewetried.Protocolsforyeastimmunofluorescencecanbefoundhere,orinMethodsinEnzymology194(1991).

  1. TransformstrainwithpB227/GAL-cre,selectingonSC-leu.
  2. ToderepresstheGALpromoter,inoculatetransformantsinto2mlsSC-ura-leuwith2%raffinoseascarbonsourceandgrowtosaturation.
  3. Dilute1/100intoSC-leuwith2%galactoseascarbonsource(control:SC-leuwith2%glucoseascarbonsource).Growfor2days(somestrainsinducewithoutgrowing).
  4. Ifgrown,dilute1/100.Spota10uldropontoanFOAplateandstreakitforsingles(non-quantitativeapproach!).OrplatedilutionsontoSCmediaandreplicatoidentifyura-colonies.Theinducedculturesshouldgive100xmoreura-cellsthanthecontrol.
  5. PCRprimersdesignedusingthesequencegivenbelowcanbeusedtodeterminepositionofthetag.TheIRelementsandpalindromicloxRregionshouldbeavoided.

N.B.Whentaggingessentialgenes,theoriginalstraintransformedshouldobviouslybediploid.Youcandissectthepopped-outversiontoseeifthetaggedgeneisfunctional.Onlybelieveatagislethalifitiscomplementedbythewild-typegene,andifseveralpopouteventsgivethesamephenotype.

SequenceofHATtag(3xHA):

TRinuppercase.loxRinbold.

GGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGgcggccattgaaggtagaagagaaaatttgtacttccaaagaaagaaggccgctatcgcttcggataactcctgctatacgaagttatgggcggccgtttacccatacgatgttcctgactatgcgggctatccctatgacgtcccggactatgcaggATCCtatccatatgacgttccagattacgctccggccgcCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCC

GenBankaccession

  • mTn-3xHA/lacZisU54828
  • pRSQ2-LEU2isU64693

Antibioticsused:

Tetracycline,Tet(SigmaT3383)12mg/mlin50%ethanol.Useat3ug/ml(Tet3)
Kanamycin,Kan(SigmaK800)10mg/mlinwater.Useat40ug/ml(Kan40)
Ampicillin,Amp(SigmaA9518)50mg/mlinwater.Useat50ug/ml(Amp50)

免责声明 本文仅代表作者个人观点,与本网无关。其创作性以及文中陈述文字和内容未经本站证实,对本文以及其中全部或者部分内容、文字的真实性、完整性、及时性本站不做任何保证或承诺,请读者仅作参考,并请自行核实相关内容。
版权声明 未经蚂蚁淘授权不得转载、摘编或利用其他方式使用上述作品。已经经本网授权使用作品的,应该授权范围内使用,并注明“来源:蚂蚁淘”。违反上述声明者,本网将追究其相关法律责任。
相关文章