TypicaltransformationresultsThefollowingtransformationswereperformedatthesametimeusingthemultiwelltransformationprotocol.SimilaramountsofPCRproductwereusedforeach.ThepicturesdemonstratethedegreeofsuccessandvariABIlitythatcanbeattained.Theplateswereincubatedfor6daysat30°Cbeforebeingphotographed.Thehaploidplateshowsthemanysmallerbackgroundcolonies(1mm)whichappearearlyduringtheincubation,inadditiontothetruetransformants(3-10mm).ThesesmallercolonieswillnotshowresistancetoG418whenrestreakedanddonotappearinthediploidstrain.Colonysizedependstosomeextentonthenumberofpositivesontheplate,ontheamountofbackgroundandonwhetherthedipruptedgenewasimportantforviability.Thetransformationefficiencyofthediploidstrainisapproximatelyone-thirdofthehaploidstrainforunknownreasons.(OnepossIBLityisthatfewerdiploidcellsareusedinthetransformationperOD600thanthehaploid.Thisneedstobetested.)

DIPLOID-----------------------HAPLOID

TransformationresultsforORFYER138C-A.Genename=TY2AGeneproduct=TY2AproteinPreviousdisruption=unknown

ThehightransformationefficiencyobservedhereisprobablyanartifactresultingfromthefactthatmultipleTYelementsexistinthegenome.SimilarresultshavebeenobservedwithcertaintelomericopenreADIngframes.Thedeletionprimerpickingalgorithmshavebeenupdatedtoensurethattargetingprimersareuniquewithinthegenome.Ifprimersarenotunique,thenlongerstretchesofhomologoussequencewillbeusedintheprimerdesign.Thisenhancementhadnotbeenimplementedfortheopenreadingframeswhichweredisruptedinthetrialstage.

DIPLOID-----------------------HAPLOID

TransformationresultsforORFYER140WGenename=unassignedGeneproduct=hypotheticalproteinPreviousdisruption=unknown

DIPLOID-----------------------HAPLOID

TransformationresultsforORFYER141WGenename=COX15Geneproduct=cytochromeoxidaseassemblyfactorPreviousdisruption=unknownThisopenreadingframemightbeagoodcandidateforanessentialgene,basedontransformationresults.Recentdata(Smithetal.,Science2742069-2074),however,suggeststhatthisorfmaybedisrupted,butresultsinastrainwithareducedgrowthrate.Therefore,itwillbeimportanttopickbothbigandsmallcoloniesforconfirmationPCR.

DIPLOID-----------------------HAPLOID

TransformationresultsforORFYER142CGene=MAG1Geneproduct=3-methyladenineDNAglycosylasePreviousdisruption=viable

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