影响电转化效率的几个因素探讨

1、1,2,3,1,1,1,2,1,1,2,1,2(1./,101300;2.,100101;3.,325027)[]:DNA::2.12.5kV;,DNA,;5;,10%:,DNA,DNA[];;;[]Q785[]A[]1000-2138(2003)01-009-03Discussiononsomefactorsaffectingelectro2transformationefficiencyBAOQi2yu3,SUNYong2qiao,WANGHui2feng,etal.3BeijingGenomicsInstitute/GenomicsandBioinformaticscenter,ChineseAcademyofSciences,Beijing101300Abstract:Objective:Toinvestigatesomefactorsaffectingelectro2transformationefficiencyinlargescaleDNAse2quencingplatform.Methods:Electro2transformationefficiencyofthel。

2、igationmixwasdetectedwhentheligationmixwastreatedwiththemethodsofwaterdilutionorethanolprecipitation.Results:Ahighelectro2transformationefficiencywasob2tainedwhenthevoltagewassetbetween2.1to2.5KV;ethanolprecipitationwouldreducethesaltconcentrationbutitcouldnotincreasetheelectro2transformationefficiencybecauseofthelossoftheplasmidduringtheethanolprecipitation;waterdilutionincreasedtheelectro2transformationefficiencybyaboutfivetimes;theelectro2transformationefficiencywashigherwhenthecompetentcells。

3、uspendedindeionizedwaterthanthatin10%glycerol.Conclusion:Lowersaltconcentra2tioninthesamplegotahigherelectro2transformationefficiencyandasimpleandefficientmethodforpurificationoftheliga2tionmixshouldbesetupforthelargescaleDNAsequencing.Keywords:electro2transformation;transformationefficiency;dilution;ethanolprecipitation,[1],,()DNA(RNA),,DNA,,,11.1BACDNApUC18E.coliDH5;pUC18DNASTRATAGENE(CAT#200231LOT#128);50(64)BAC(),(5064)BAC:2001-12-17:(1961-),,,,DNA(,BAC),,-701.2[2]1%LB,37(23h)OD6000.60.8,4,,。

4、10%,21011/ml,100l,-701.3[2]BIO2RADE.coliPulser,100l,1mm,1.52.5kV,1l100l,(1mm)AmpLB,37,3,5%1.41933120032JournalofWenzhouMedicalCollegeVol.33No.1Feb.2003©1994-2008ChinaAcademicJournalElectronicPublishingHouse.Allrightsreserved.(pH5.0),2,,-7020min,413000r/min20min,,70%,,,1DNA22.11.52.5KV,1l0.01ngpUC18DNA1l50BACDNA(pUC18),12.1KV2.3KV2.5KV,1.5KV1.7KV1.9KV,50BACDNA10%1Tab.1Relationshipbetweenvoltageandelectro2transformationefficiencyvoltage(KV)electro2transformationresult(clones)0.01ngpUC18DNAinwater(。

5、1l)50BACDNAsubclonelibrary(1l)10%glycerol3water3310%glycerol3water331.54.51031.31041.41042.61041.76.01031.31043.31048.41041.92.01044.61044.61048.21042.19.21048.31041.41052.41052.37.51049.41048.21042.31052.59.01046.21048.81041.81053:competentcellsuspendedin10%glycerolwater;33:competentcellsuspendedindeionizedwater2.2pUC18DNA1,,22.1KV,1l0.01ng0.001ngpUC18DNA1,1101002(2.1KV)-1Tab.2Effectofligationbufferonelectro2transformationefficiency(2.1KV)-1electro2transformationresult(clones)310%glycerolwater3。

6、water330.01ngpUC18DNAinwater(1l)2.110103332.410103330.01ngpUC18DNAin1ligationbuffer(1l)5.21083331.61093330.001ngpUC18DNAinwater(1l)3.310103332.610103330.001ngpUC18DNAin1ligationbuffer(1l)1108333110833350BACDNAsobclonelibrary(1l)1.31051.91053:competentcellsuspendedin10%glycerolwater;33:competentcellsuspendedindeionizedwater;333:clonesperugpUC18DNA,,1105,33(2.1KV)-2Tab.3Effectofligationbufferonelectro2transformationefficiency(2.1KV)-2volume(l)electro2transformationresult30.01ngpUC18DNAin1ligationb。

7、uffer64BACDNAsubclonelibrary10%glycerolwater33water33310%glycerolwater33water3331.06.01081.11091.51042.21040.71.81093.01094.61045.51040.52.61094.61096.01048.01040.33.41095.61097.41048.31040.13.51095.81097.61048.71040.001ngpUC18DNA/1lwater1.310108.910103:clonesperugpUC18DNAorperul60BACDNAsubclonelibraryligationmix;33:competentcellsuspendedindeionizedwater;333:clonesperugpUC18DNA2.3DNA0.01ngpUC18DNA(1ligationbuffer,1l)50BACDNA(1l),4,pUC18DNA1.6104();,1102,10050BACDNApUC18,1.9105;,3.2105(),4Tab.4Ef。

8、fectof70%ethanolprecipitationofsamplesonelectro2transforma2tionefficiency(2.1KV)electro2transformationresult(clones)0.01ngpUC18DNAin1ligationbuffer(1l)50BACDNAsubclonelibrary(1l)10%glycerolwaterwater10%glycerolwaterwaterpre2precipitation5.21031.61041.31051.9105post2precipitation110211022.31053.21053DNA106108/gDNA,109[24]1010DNA,0.010.1ngDNA,0.01331©1994-2008ChinaAcademicJournalElectronicPublishingHouse.Allrightsreserved.[4],1l0.01ng0.001ngpUC18DNA1,2,[5,6][5,6]DNA,DNA1l0.01ng0.001ngpUC18DNA1,(10。

9、100);50BACDNApUC18,;10700[5],,,;10[6],64BACDNA0.01ngpUC18DNA110,50.01ngpUC18DNA10DNA,DNA:[1]EricS.Lander,LaurenM.linton,BruceBirren,etal.Initialsequenc2ingandanalysisofthehumangenome[J].Nature,2001,409:860-921.[2]F.,RE,JG,.,,.[M].:,1998.23-24.[3],.DNA[J].,1999,9(4):5-8.[4]WilliamJDower,JeffFMiller,CharlesWRagsdale.HighefficiencytransformationofE.colibyhighvoltageelectroporation[J].NucleicAcidsRes,1988,16(13):6127-6145.[5]MoniqueJacobs,StephanWnendtandUlfStahl.High2efficiencyelec2tro2transformati。

10、onofEscherichiacoliwithDNAfromligationmixtures[J].NucleicAcidsRes,1990,18(6):1653.[6]TracyAWillsonandNicholasMGough.HighvoltageE.colielectro2transformationwithDNAfollowingligation[J].NucleicAcidsRes,1988,16(24):11820.(:,)(,325027)[];;[]R683.41[]B[]1000-2138(2003)01-0011-01,,54,,,:30,;,(+),(+),,;,(+),;,X:150,,;,:,,,,,,,;,,;903w,;2w,2w2:2002-09-28:(1964-),,,,:,,,;,:150,,,,,;,1/3,,,,,,,:,,,,,,,,(:)1133,:1©1994-2008ChinaAcademicJournalEl。