酵母转化问题参考.docx

酵母转化问题参考

第一篇：酵母转化问题参考WaystoDestroyYourYeastTransformationbyEmilyCrowon27thofJuly,2011inCell/TissueCultureTransformingyeastwithDNAisaverysimilarprocesstotransformingE.coli,butwithjustenoughdifferencestotripyouupifyouletyourattentionslip.Whetheryou’redoingayeasttwo-hybridscreen,orusingyeastasamodelsystem,hereareasomemistakestotoavoid…1.ForgettingtoaddsinglestrandedDNAWhileE.colireadilytakesupdouble-strandedDNA,yeastrequirestheadditionofsingle-stranded“carrierDNA”toenhanceuptakeofyourplasmidorfragment.Ifyouonlyaddyourplasmidtothetransformationmix,chancesareyou’llbeconfrontedwithapristinelysterileplateafterthreedaysofincubation.2.UsingoldPEGPEG(polyethyleneglycol)isacrucialingredientintheyeasttransformationbuffer.Unfortunately,it’sannoyingtomakeandgoesbadquickly.ThepercentageofPEGwillmakeorbreakthesuccessofyourtransformation;anoldPEGsolutionhashadachancetoevaporate,sothatthewatertoPEGratioisoff.Ifyoucanstandit,makenewPEGforeverytransformation.Otherwise,makeitinsmallbatches,andsealtightlywithparafilmbetweenusestominimizeevaporation.3.UsingcellsinstationaryphaseCellsinlogphase,orexponentialgrowth,takeupDNAwithmuchbetterefficiency.Thisisnottosaythatcellsfromastationaryculturecan’tbetransformed–onlythatyoursuccessfultransformationratewillbemuchlower.Yourbestbetistousecellsthataregrowingrapidlyatthetimeoftransformation.4.UsingthewrongselectivemarkerAstupidmistake,butonethatI’vemademoretimesthanI’dliketoadmit.Doublechecktosaveyourselfsometears!5.Cuttingcornerswhenheat-shockingThisisamajordifferencebetweenE.coliandyeasttransformations:whileE.coliisrelativelydelicate,andrequiresaheat-shockoflessthanaminutetotakeupDNA,thecellwallmakesyeastmorehardyandresistanttoshock.Thus,forefficienttransformation,yeastaregenerallyheat-shockedforupto45minutes.I’vegottenawaywithaslittleas15minutes,butanyshorterandthetransformationefficiencyisdrasticallyreduced.Ifyou’renotinahurry,letitgothewhole45minutes,oryou’llgettoexperiencethejoyofdoingitalloveragain.第二篇：关于酵母类产品是否上税问题根据相关法律来分析，酵母细胞壁多糖和酵母自溶物