预览加载中，请您耐心等待几秒...

质粒提取与酶切电泳实验报告.doc

预览
1/7
2/7
3/7
4/7
5/7
6/7
7/7

在线预览结束，喜欢就下载吧，查找使用更方便

下载提示 文本预览

如果您无法下载资料，请参考说明：

1、部分资料下载需要金币，请确保您的账户上有足够的金币

2、已购买过的文档，再次下载不重复扣费

3、资料包下载后请先用软件解压，在使用对应软件打开

PAGE\*MERGEFORMAT7 分子实验报告2013030020华天瑞 PreparationofPlasmidDNA,RestrictionEnzymeDigestion,and AgaroseGelElectrophoresis 2014/10/14-21 1Intro 1.1Objective Tolearn ThecharacteristicsofplasmidDNA ThemethodofplasmidDNAmini-preparationbyalkalinelysisandthemeasurementofDNAconcentrationbyspectrophotometer Thecharacteristicsofrestrictionendonuclease HowtouseagarosegelelectrophoresistoseparateDNAs Tounderstand: TheprinciplesofpurificationandquantificationofplasmidDNA 1.2Principle 1.2.1PlasmidandVector Plasmidisasmall,independentlyreplicating,pieceofextrachromosomalcytoplasmicDNA(doublestrandedandusuallycircular)thatiscapableofautonomousreplicationandcanbetransferredfromoneorganismtoanother. VectorserveascarrierstoallowreplicationofrecombinantDNAinthehostcell,usuallyavectorcovers Antibioticresistancegene:suchasampicillinresistantgene,kanamycineresistantgene,andetc. Originofreplication(ori). Multiplecloningsite(MSC)orpolylinker Markergenes:suchasLacZgene. 1.2.2AlkalineLysis(0.2molNaOH+1%SDS) SDSisakindofanionicdetergent.Itcanbreakbacterialcellsanddenatureproteins.Whenbacterialcellwallisbroken,theplasmidDNAandgenomicDNAwillbereleasedoutandbedenaturedinalkalienvironment.Whenthesolutionisneutralizedbyacidicreagent(suchasKAc),theplasmidDNAwillberenaturedrapidlyduetoitssmallersize.Aftercentrifugation,theplasmidDNAwillbeinsupernatant,whilethegenomicDNAwillstayinthesedimentatthebottomofthetubestogetherwithothercelldebris. 1.2.3DNAConcentrationMeasurement Basedonthestrongabsorbanceofbasepairs(A-T,G-C)at260nmUV,theconcentrationofDNAcanbemeasuredbyspectrophotometry.Whendetectedunderneutralcondition,A260isusedtocalculatethenucleicacidconcentrationwhereastheratioofA260/A280canbeusedtoestimatethepurityofnucleicacid(1.8forpureDNA). 1.2.4RestrictionEndonuclease TypeIIREcutsdsDNAatspecificrestrictionsitesonspecificsequence,producingrestrictionfragments. 1.2.5GelElectrophoresis Solidifiedagarosesolutionhascertainsizeofsmallporesofwhichthesizeisdecidedbyconcentration.Intheelectricfieldand