双荧光素酶报告基因检测系统-promega

Dual-Luciferase®ReporterAssay试剂准备TOC\o"1-5"\h\zLuciferaseAssayBufferII10mlLuciferaseAssaySubstrateIvialStop&Glo®Buffer10mlStop&Glo®Substrate,50X200ulPassiveLysisBuffer（PLB）,5X30ml1XPLB:加1体积的5XPassiveLysisBuffer（PLB）到4体积的dH0中，机保存（一个月）。2LARII：将LuciferaseAssaySubstrate重悬于10mlLuciferaseAssayBufferII中（-200。保存1个月，-70oC保存1年）。1XStop&Glo试剂：1体积50XStop&Glo®Substrate加入49体积的Stop&Glo®Buffer中（-20oC保存15天）。（每次试验需要100ul）细胞处理吸除细胞培养液1XPBS轻柔的冲洗细胞加入1乂PLB（推荐用量）Volumesof1XPLBtoUseInStep3.PassiveLysi§ActiveLy&isPlateSize1XPLBDis^PlateSize1XPLB6-well50如I100x200mm1ml12-well250pl60x15mmtopi24-well10叩35x12mm200pl48-well65pl6-W&II25印I96-well20pl12-welllOOul4.细胞溶解室温条件下，轻摇细胞15min，cellL暨信Feme岫growlhmedaIronicell*RinswllliIXPBS./一1KFLB.Perloim御s.Translertoanewtubewvial.-

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injeclirmminaieier|insidglurnnamtiBrjPlate"I〕_20p|匕"脚射岫1。削Ispen踌Id卯I计UWIIntnlumlnonetertuts.Pro-amLiiingler.DiSCelEti100^01LAfill.MeasureInllyluciferaseaclhlty.DispeiEfl10卯lolSlop&GICl®磁网LMeasureR^iUalucifiefaseactivity.hleasu矿Irpflyluciferase汨iwily.曲piWfl1祯IalStop&G睛Raagenl.mn血r20plofPLBL牌胡.MIX.RepealcyclelorromatilngwNsinplale.sn'=mEI号MeasureRet艰总l此imra推activity.3AdditfcnalpralocoIinformalionismailableinTechnicalManual打船。ci彻傩availableonlineat；www.pmmsi|3.com瞬时转染和报告基因实验采用脂质体介导技术转染。重组质粒分别为p-629/+100,p-401/+100,P-238/+100，p-80/+100，p-25/+100。pGL3-basic为阴性对照；同时以转染phRL-tk(海肾荧光素酶)作内对照。具体转染方法参照转染(PolifectamineReaent)说明书进行。将质粒DNA(3.2pg)与phRL-tk(0.8pg)按1:4混合后为A液，混匀30s，PolyFect(QIAGEN)与无血清无抗生素的DMEM按1:50混匀后为B液，混匀30s;A+B混匀^加入A)15s，室温下孵育5-10min;吸出六孔板中的培养液，用无血清无抗生素的DMEM洗3遍，然后加入AB混合液，每孔0.8mL;6h后，加入2mL完全DMEM;24h后，倒出旧培养液，换为完全DMEM;100ugHO或B(a)P处理lh或24h;(200uMMMS，24h-溶解于MeSO,Sigma)；22、、、、2(H2O2不引起OGG1升高)采用Promega公司的