ProtocolforYeastRecombinationalCloning

AmapofacapturevectorusedtoclonegenomicDNAinyeastisdepictedinfigure1.
Constructionofthecapturevector:
Thecapturevectorisassembledusinghomologousrecombinationinyeast.Thereare
fourfragmentsthatareneeded:upstreamanddownstreamtargetingsequences
homologoustothebordersofthedesiredfragmenttobecloned(1kbeach)generatedby
PCR,thevector,pLLX13,thatcontainscomponentsnecessaryfor1)thecapturevector's
selectioninyeast,E.coli,andP.aeruginosa,2)replicationinyeastandbacteria,and3)its
transfer,andaPCRproductamplifiedfrompLLX8containinganampicillinresistance
geneandacounterselectionagainstthegrowthofyeast(CYH2)inthepresenceof
cycloheximide.
Alternatively,pLLX16KancanbeusedinsteadofpLLX13.Thisvectorisnotableto
replicateinP.aeruginosa.
Adiagramofthefourfragmentsandhowtheywillrecombinetoformthecapturevector
isincludedinfigure2.
Primerdesignforthetargetingsequences:
Theupstreamanddownstreamtargetingsequencesthathavebeenusedareonekbeach
inlengthandaredesignedtobethesequencesborderingtheDNAregionthatisintended
tobecloned.TheyaregeneratedbyPCRandhomologiestofacilitaterecombination
necessaryforassemblyofthecapturevectorareaddedtotheupstreamanddownstream
targetingsequencesbyaddingtailstothe5'endofeachprimer.Fortheupstream
targetingsequence,theextensiontothesenseprimer,namedP1,is:5'ATATTACCC
TGTTATCCCTAGCGTAACTATCGATCTCGAG3'.Thissequenceprovides40
bpofhomologytopLLX13.
Thetailaddedtotheantisenseprimer,namedP2is:5'CATATATACTTTAGATTT
TAATTAAACGCGTTCTAGAAAA3'Thissequenceprovides40bpofhomology
tothepLLX8PCR.
Forthedownstreamtargetingsequence,thetailaddedtothesenseprimer,namedP3,is:
5'CATTTTCACCGTTTTTTGTTTAAACGTTAACTCTAGAGGG3'This
sequenceprovides40bpofhomologytothepLLX8PCR.
Thetailaddedtotheantisenseprimer,namedP4,is:5'TAACAGGGTAATATAGAG
ATCTGGTACCCTGCAGGAGCTC3'thissequenceprovides40bpofhomologyto
pLLX13.

Preparationofthefragments:
PCRtheplasmidpLLX8withtheprimers:5'TTTTCTAGAACGCGTTTAATT
AAAATCTAAAGTATATATGAGTAAAC3'
and
5'CCCTCTAGAGTTAACGTTTAAACAAAAAACGGTGAAAATGGGT
GATAG3'
theresulting2.95kbfragmentwillcontaintheblaandCYH2genes
Togeneratetargetingsequences,usethetwosetsofprimersdesignedaccordingtothe
instructionsabove.
GelpurifythePCRproducts.
LinearizetheplasmidpLLX13DNAwiththeenzymeNheI:
resultingina9.940kbfragment.
Heatkilltheenzymefromthedigest.
Youwillneedapproximately200ngeachoftheupstreamanddownstreamhomology
fragments,600ngofthepLLX8PCRand200ngofthepLLX13digestper
transformationofyeast.Theseamountsdonotneedtobeexact.Theassemblyisusually
veryefficient.
ThenextstepistocotransformallfourofthefragmentsintocompetentS.cerevisiae
strainCRY12whichisresistanttocycloheximide.
Transformationprotocol:
StreakontoaYPDplatestrainCRY12.Thiscanbereusedfor12months.
Inoculateafewcoloniesinto50mlofYPD.Growina250mlbaffledflaskshakingat250
rpmat30degreesCelsiusovernight.After1216hrsinoculatethedesiredamountof
YPD(every50mlgivesapproximately1.5transformations)withadilutionof
approximately1:100andadjustthefinalOD600soitisapproximately0.05.Letthe
culturegrowforaroundfivehourswithshakingat30degreesuntilthefinalODis0.4
0.5.Transferthecultureto50mlconicaltubesandcentrifugeatroomtemperaturefor5
minutesat1,200rcf.
Discardthesupernatantandresuspendin40mlsterileRTwater.Spinagainatroom
temperaturefor5minutesat1,200rcf.

Discardthesupernatantandresuspendin250microlitersTFBI.AddtheDNAtoa5ml
snapcaptubealongwith200microgramsofsinglestrandedherringspermDNA.
pLLX13shouldbetransformedasapositivecontrol.Thenadd200microlitersofthe
yeastsuspendedinTFBItothe5mltubefollowedby1.2mlofTFBIIandvortexuntil
homogeneous.
Putthetubesina30degreewaterbathfor30minutesfollowedbya42degreewaterbath
for15minutes.Thenpelletthecellsfor2minutesat1,200rcf,dumpthesupernatant,and
gentlyresuspend(novortexing)in500microlitersTE.Plate100microlitersontouracil
dropoutplatesandputina30degreeincubator.Colonieswillappearin23days.There
shouldbehundredsofcoloniespertransformation.
Toobtainthedesiredplasmid,purifyDNAfromthetransformantsasapool.Swabeach
plate,resuspendtheyeastinPBS,pelletthecellsandthenuseaZymoprepyeastplasmid
miniprepkittopurifytheyeastDNA.ThenelectroporateintoE.coliDH10B(high
efficiencyisrequired)selectingfor5micrograms/mltetracylineand100micrograms/ml
ampicillinresistanceifpLLX13wasusedor50micrograms/mlkanamycinand
ampicillinifpLLX16kanwasused.Pickcoloniesandanalyzeforthedesiredplasmid.
Afterselectiononampicillinandtetracyclineorkanamycin,usuallygreaterthan90%of
theplasmidsanalyzedarecorrect.
Whenthecorrectplasmidhasbeenidentified,performatesttransformationofyeastto
verifythecycloheximidecounterselectionworks.Selectonbothuracildropoutmedia
withandwithoutcycloheximide2.5micrograms/ml.Therateofreversionfor
cycloheximidesensitivityshouldoccurinlessthan1%ofthetransformants.Itisaround
10percentforlinearizedplasmids.
RecombinationalCloning:
LinearizethevectorDNAwithPmeIorMluI.
IfthesizeofthepieceofDNAtobeclonedislessthanorequalto30kb,thenthe
previousmethodofpreparingyeastcompetentcellsisrecommended.Pertransformation,
use500ngoflinearizedvectorand5microgramsofshearedgenomicDNA(passed
througha261/2gaugeneedle40times).
Transformthecapturevectorasapositivecontroltotestthetransformationefficiency.
SelectforcapturevectorscontaininggenomicDNAbyplatingthetransformationon
uracildropoutplateswiththeadditionofcycloheximide.Expect0to100coloniesper
transformation.
Inmyexperience,2%to10%ofcolonieshaveplasmidswiththegenomicDNAthatwas
desiredtobecloned.

IfthesizeoftheDNAtobeclonedisgreaterthan30kb,thenthespheroplastmethodof
preparingcompetentyeastmaybetheonlywaytoclonetheDNAofinterest.Iamnot
sureaboutthisbutfromthelab'sexperience,30kbfragmentsareeasilyclonedusing
chemicallycompetentyeastbutfragments85kbinlengthhaveneverbeensuccessfully
clonedusingchemicallycompetentyeastandhaveonlybeenclonedbyusing
spheroplasts.
Transformationprotocolforpreparingspheroplasts:
Innoculate50mlofYPDasdescribedbefore,dilute1:100andgrowtoOD600of0.5to
0.8.Transferthecultureto50mLconicaltubesandspinat1,200rcffor5minutesat
roomtemperature.
Washonetimein40mLssterilewaterandcentrifugeasabove.
Repeatusing20mLs1Msorbitol.
Resuspendin20mLsSCE.
Remove100microlitersforfutureuse.
Add200microliters1MDTTand50microlitersZymolyase20T(10mg/mLin10mM
sodiumphosphatebufferpH7.5)
Placetubesina30degreewaterbath.
Monitorforspheroplastformationbyadding100microlitersofyeastto900microliters
ofwaterandcomparetheOD800readingtothenonspheroplastsamplethatwas
removedearlier.WhentheOD800readingis10%oftheoriginal,removefromthewater
bath.
Atthispoint,itisnecessarytobemuchmoregentlywiththecellsbecausetheyaremore
fragileduetothedigestionofthecellwall.
Centrifugeat300rcffor4minutesatroomtemperature.
Resuspendin20ml1Msorbitol.
Washasaboveusing20mlSTC.
Resuspendin2mlSTC.
Pertransformationgentlymix200microlitersyeast,1microgramlinearizedplasmid,10
microgramsofunshearedgenomicDNA,and5microgramsofsinglestrandedherring
spermDNAalltoa5mlsnapcaptube.

Alwaysincludeapositivecontrolforthetransformationsuchasthecapturevectoror
pLLX13.
Letsitatroomtemperaturefor10minutes.
Add2mlPEG,gentlymix,andincubatefor10moreminutes.
Centrifugeat300gfor5minutesandgentlyresuspendin150microlitersSOS.
Incubateat30degreesfor30minutes.
Addto8mltopagarat46degreesandpourontoa1Msorbitoluracildropout
cycloheximide2.5micrograms/mlplate.
Coloniesfromthepositivecontroltaketwodaystoappear.Coloniesfromthe
recombinationalcloningcantakethreedaystoappear.
ToScreencoloniesforcorrectinsert:
Patchfirstontothesameselectivemediaaspreviouslyused.Thisisnecessarybecause
youcangetfalsepositivesfromthePCRscreeningbelowifthecolonyisn'tpatched
beforeusingforPCRduetoresidualgenomicDNAontheplate.
Createspheroplastssothecellsmaybelysed:
Resuspendasmallamountofyeastfromeachpatchinto1015microlitersspheroplast
solution.
Incubateat37degreeswithshakingfor1hr.
PCRtodetectplasmidscontaininggenomicDNA:
Add1microlitersoftheyeastspheroplastsintoa25microliterreaction.
ForthePCRprogram,incubateforfiveminutesat94degreescelsiusanduse40cycles
ofdenaturing,annealing,andextension.
DesignyourowncontrolsforyeastlysisandPCRareveryimportantasthisstepisprone
toerror.
Uponidentifyingthepatchcontainingtheplasmidwiththecorrectinsert,resuspendthe
patchinPBSandpellet.IsolateDNAaspreviouslydescribedandelectrotransformone
microliterintoE.coliDH10B.SelectonLAgarwith5micrograms/mltetracycline
(pLLX13)or50micrograms/mlkanamycin(pLLX16kan).

Uponobtainingcolonies,performanotherPCRtoverifythatthedesiredplasmidisstill
present.
TopurifyplasmidDNA,performaQiagenMIDIorMAXIprep.

SolutionsandMedia:
TFBI
1volume1MLiAcetate
1volume10XTEpH7.5
8volumeswater
TFBII
1volume1MLiAcetate
1volume10XTE
8volumes50%PEG3350(Sigma)
UracilDropoutPlates/Liter
20gagar
800mlwater
Autoclaveand
Add200mlsterile5X(perliter)MinimalSDmedia(BDBiosciencesClontechCat#
630411/86021)+UracilDropoutsupplement(ClontechCat#86071)
TopAgar
2.5%Agar
1MSorbitol

STC
1MSorbitol
10mMtris,pH7.5
10mMCaCl2
SCE
1MSorbitol
100mMNaCitratepH5.8
10mMEDTApH8

PEG
20%PEG8000(Sigma)
10mMTrisbase(pH7.5)
10mMCaCl2
SOS
25mlYPD
700microliters1MCaCl2
18.2gsorbitol
waterqsto100ml
Spheroplastsolution:
MakeastockconcentrationofZymolyase20T25mg/mldissolvedin10mMsodium
phosphatebufferpH7.5and1Msorbitol.Freezeat80Cin100microliteraliquots
Dilute1:10inthesamesolutionitwasdissolvedinforusethatcontains10mMDTT.

Supplies:
Zymolyase20T:
ICNcat#320921
Cycloheximide:
ObtainedfromSigma,cat#C7698
Stocksolutionis10mg/ml
Dilute1:4000tomake2.5micrograms/ml
SinglestrandedherringspermDNA:
Clonetechcat#K1606A(yeastmakercarrierDNA)
Uponreceipt,boilfor5minutesandthenplaceonicetoensureitissinglestranded,and
thenaliquotintoappropriateamounts
Zymoprep,YeastPlasmidMiniprepkitisobtainedfromZymoResearchcat#D2001
ElectrocompetentE.coli:
InvitrogenelectrocompetentE.coliDH10B18290015
InvitrogenhasavariantofDH10BcalledGeneHogs(cat#C8080)thathavebeen
modifiedtotakeuplargeplasmids.