pMD2.G慢病毒载体使用说明

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1、pMD2.G编号载体名称北京华越洋VECT23114pMD2.GpMD2.G载体基本信息：载体名称:pMD2.G哺乳动物载体；慢病毒包装载体；双质粒包装系统；信质粒类型:封载体高拷贝/低拷贝:高拷贝克隆方法:限制性内切酶，多克隆位点启动子:CMV载体大小:5824BP5'测序引物及序列:CMVfwd5’CGCAAATGGGCGGTAGGCGTG3’3'测序引物及序列:--载体标签:无载体抗性:Ampicillin筛选标记:无克隆菌株:DH5α或HB101宿主细胞（系）:包装细胞系如293T慢病毒包装载体pMD2.G是2质粒包装系统的信封质粒备注:与psPAX2一起使用

2、，适用范围、使用方法见下文。也可以与第三代包装载体pRSV-Rev、pMDLg/pRRE一同使用，构成3质粒包装系统。稳定性:瞬表达组成型/诱导型:组成型病毒/非病毒:非病毒pMD2.G载体质粒图谱和多克隆位点信息：pMD2.G载体简介：第二代慢病毒包装质粒pMD2.G使用方法-慢病毒包装与转染方法Day11.Plate5x105293Tcellsin6cm2dishescontaining5mLofmedia.(Thiscanbescaledupifdesired)Day21.Setup(usepolypropylenetubesforthis;polystyren

3、etubesDONOTwork!):1µgretroviralDNAencodinggeneX1µgpackagingplasmidMixthepackagingplasmid(psPAX2)ata8:1ratiowiththeenvelopeplasmid(pMD2.G)-atotalof1µgDMEwithoutserumto94μLtotal6μLFugeneMixandwait15to30minutesatroomtemperatureAddto293Tcellswithouttouchingthesidesofthedish(DONOTCHANGEMEDIA

4、)IfyouareusingamphotropicvirusthenmoveimmediatelytoBL2+inasecondarycontainer,whichhasanabsorbentmaterial.(Thisdoesnotmeanacoupleofhours;itmeansImmediately!).Therestofthisprotocolisthesameforallviruses---theBL2+safetypracticesareinplaceifyouareusingamphotropicvirusesDay31.Changethemediat

5、owhatevermediayouwishtousewheninfectingtargetcells.293Tcellsareeasilydetachedsoremembernottoputthemediadirectlyontotocells,butrather“run”itdownthesideofthedish.Rememberthatyouwillgetthehighesttiterviruswhenyourcellsare“happy.”2.PlateoutyourtargetcellDay41.Removethemediumfromthe293Tcells

6、andusea0.45usyringefiltertoremoveany293Tcells.DONOTusethe0.2ufilter,asitislikelytosheartheenvelopefromyourvirusmakingitnoninfectiousNote:Afterfiltering,thefiltershouldberemovedandplacedinthebiohazardbaginthehoodandthesyringerinsedwithbleachanddecontaminatedforaminimumof20min.Itisusefult

7、oplaceaplasticbeakerwithbleachinthehoodinadvance2.Add8to10μg/mlofpolybrene(Hexadimethrinebromide)orprotaminesulfatetothetargetcells3.Carryoutinfectionfor1to4hours.RemovevirusandreplacewithfreshmediaNote:Ifyouwishtodoasecondinfectionthefollowingday,itisimportanttoputfreshmediaon