轉(zhuǎn)化簡(jiǎn)短發(fā)展史和基礎(chǔ)實(shí)驗(yàn)流程課件

ABriefHistoryofTransformationand

Basic

ProtocolsOctober17,20162022/12/2511.IntroductionImage’scopyrightbelongtoWikipediaThisprocessofbacterialcell2(thesquareone)takingupthenewgeneticmaterialwhichisbelongtobacterialcell1(theellipticalone)iscalledTransformation.2022/12/2522.BriefHistoryFredGriffith.Thesignificanceofpneumococcaltypes[J].Hyg,1928,

27:113–159.

LederbergJoshua

.TheTransformationofGeneticsbyDNA:AnAnniversaryCelebrationofAVERY,MACLEODandMCCARTY(1944)in

Anecdotal,HistoricalandCriticalCommentariesonGenetics,1994,TheRockefellerUniversity,NewYork,NewYork10021-6399.格里菲斯肺炎雙球菌轉(zhuǎn)化實(shí)驗(yàn)：“轉(zhuǎn)化因子”奧斯瓦爾德·埃弗里肺炎鏈球菌轉(zhuǎn)化；DNA為遺傳物質(zhì)2022/12/2542.BriefHistory大腸桿菌在CaCl2作用下吸收噬菌體“λ”CohenStanleyN,ChangAnnie,HsuLeslie.NonchromosomalAntibioticResistanceinBacteria:GeneticTransformationofEscherichiacolibyR-FactorDNA[J].ProceedingsoftheNationalAcademyofSciences,1972,69(8):2110–4.大腸桿菌在CaCl2作用下有效轉(zhuǎn)化質(zhì)粒MandelMorton,HigaAkiko.Calcium-dependentbacteriophageDNAinfection[J].JournalofMolecularBiology,1970,53(1):159–162.DouglasHanahan.StudiesontransformationofEscherichiacoliwithplasmids[J].JournalofMolecularBiology,1983,166(4):557–580.1、目標(biāo)菌株置于0℃冰浴進(jìn)行孵育；2、二價(jià)金屬陽離子（Mg2+、Ca2+）對(duì)轉(zhuǎn)化有促進(jìn)作用；3、二甲基亞砜、二硫蘇糖醇及烏洛托品鈷的作用；4、轉(zhuǎn)化效率隨質(zhì)粒的增大呈線性遞減。CaCl2作用下的質(zhì)粒轉(zhuǎn)化規(guī)律及條件的優(yōu)化IF：4.517（15-16）2022/12/2552.BriefHistoryENeumann,MSchaefer-Ridder,Y.Wang,P.H.Hofschneider.Genetransferintomouselyomacellsbyelectroporationinhighelectricfields[J].TheEMBOJournal,1982,1(7):841–5.電穿孔法增加細(xì)胞膜（鼠）的通透性，可使大分子物質(zhì)進(jìn)入細(xì)胞；但首批實(shí)驗(yàn)產(chǎn)生的穩(wěn)定轉(zhuǎn)化子并不多。EMBO：EuropeanMolecularBiologyOrganization(IF:9.643)（15-16）2022/12/2562.BriefHistorySugar,IstvanP.;Neumann,Eberhard.Stochasticmodelforelectricfield-inducedmembraneporeselectroporation[J].BiophysicalChemistry,1984,19(3):211–25.電場(chǎng)下細(xì)胞膜電穿孔法的隨機(jī)模型IF：2.363（15-16）2022/12/257PotterHuntington,HellerRichard.TransfectionbyElectroporation.CurrentProtocolsinMolecularBiology[M].2003.CHAPTER:Unit–9.3.2.BriefHistory《電轉(zhuǎn)化實(shí)驗(yàn)流程》，刊載于03年出版的《現(xiàn)行分子生物技術(shù)通用指南》，流程共13頁，詳細(xì)介紹了電轉(zhuǎn)化實(shí)驗(yàn)的各個(gè)過程，適用于絕大多數(shù)細(xì)胞（側(cè)重于哺乳動(dòng)物細(xì)胞及植物細(xì)胞）?；A(chǔ)實(shí)驗(yàn)流程包括：1Materials2Preparethecellsforelectroporation3AddDNAandelectroporatethecells4Cultureandharvestthetransfectedcells2022/12/2593.BasicProtocolsBasicprotocolofTransformationNote:AddgeneownsthepictureSpeciesStepsE.coliJM105【1】E.Coli【2】

StrainspreparationBacteriaweregrowninliquidmediaat37°Ctomid-logphase(opticaldensityof0.3at600nm)withshaking.E.coliwreGrowninLBliquidmediaat37°Cfor10-12h.;Inoculatedittonewmediainonepercent.

CompetentcellpreparationWashedonceintransformationbuffer(300mMsucrose,7mMsodium

phosphate,pH7.4,1mMMgC12)and

resuspendedintransformationbuffertoyield109colonyformingunits

(cfu)/ml.Washedtwiceintransformationbuffer(10%glycerin)andresuspendedin10%glycerinwithavolumeabout0.75%oftheinitialvolume

MixedcellswithplasmidinacertainproportionThecellsuspension(1ml)wasmixedwith200ngofpKT231-DNA,incubatedfor30minonice,analiquotwasremovedforcontrolsand800μLofthemixtureweresubjectedto

electroporation(onepulseat6250V/cmusingthe25gFcapacitor)inacooledcuvette.

Thecellsuspension(150μl)wasmixedwithabout1μgofP1000-DNA,incubatedfor15minonice;Onepulseat200Ω,25

μFd,2.5kVwithacooled2

mmcuvette.Timelastto3seconds.

IncubatedandChoosedpositivecloneTemixturewaskeptfor20minonice,dilutedintheoriginalgrowthmedium,incubated37°Cfor45minandvariousamountsplatedonoriginalmediumplatescontaining50μgkanamycin/ml.

Incubated37°Cfor1handvariousamountsplatedonoriginalmediumplatescontainingappropriateconcentrationofantibiotics[1]ReinhardWirth,AnitaFriesenegger,etal.Transforma