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普洛麦格(北京)生物技术有限公司Promega(Beijing)BiotechCo.,Ltd电话:8008108133,01058256268中文网址:英文网址:目录1.产品描述................................................................................................................................................................22.产品组分及储存条件............................................................................................................................................33.一般注意事项........................................................................................................................................................43.A光谱特性......................................................................................................................................................43.BMgCl2浓度.....................................................................................................................................................43.C兼容仪器......................................................................................................................................................44.GoTaq®qPCRMasterMix操作步骤.......................................................................................................................54.A应用ABIPRISM®7000/7300/7500/7500Fast/7700/7900HTReal-TimePCRSystem的操作方法...........64.B应用Bio-RadiQ5OpticalSystem的操作方法............................................................................................74.C应用RocheLightCyclerRealTimePCR扩增仪的操作方法.......................................................................84.D应用CepheidSmartCyclerⅡSystemRealTimePCR扩增仪的操作方法............................................104.E应用StratageneMx3000PTMRealTimePCR扩增仪的操作方法..............................................................115附录.......................................................................................................................................................................125.A快速操作指南............................................................................................................................................135.B进行RT-qPCR时的实验方法....................................................................................................................145.C实验条件的优化........................................................................................................................................165.D引物设计及使用说明................................................................................................................................175.ERealTimePCR数据分析.............................................................................................................................185.F相对定量vs绝对定量.............................................................................................................................195.G常见问题....................................................................................................................................................215.H相关产品....................................................................................................................................................221.产品描述GoTaq®qPCRMasterMix是采用染料法进行RealTimePCR的专用试剂。本产品采用一种新的专利荧光染料BRYTGreen®dye,此染料与双链DNA特异性结合后,会发出比SYBR®GreenI更强的荧光信号。用于基因定量,尤其适合低拷贝基因的检测。GoTaq®qPCRMasterMix产品提供易用的、2X的预混液。本产品中已包含:专利的双链DNA结合染料BRYTGreen®dye,低浓度的CXR(carboxy-X-rhodamine)参比荧光染料(性质与ROX相同),GoTaq®热启动酶,MgCl2,dNTP和专利的反应缓冲液。另外独立包装的100XCXR用于需要高浓度参比荧光染料的仪器。GoTaq®qPCRMasterMix的优点染料:与SYBR®GreenI染料相比,GoTaq®qPCRMasterMix专利染料BRYTGreen®dye结合双链DNA后发出的荧光更强,而且对PCR反应无抑制作用。可以让样品的CT值更早出现,线性范围更广。BRYTGreen®dye与参比荧光染料CXR的激发和发射波长分别与SYBR®GreenI和ROX相近。在实验过程中采用与SYBR®GreenI和ROX完全相同的设置。DNA聚合酶/缓冲液配方:GoTaq®热启动酶采用专利的抗Taq酶抗体,在室温时阻断Taq酶的活性。当普洛麦格(北京)生物技术有限公司Promega(Beijing)BiotechCo.,Ltd电话:8008108133,01058256268中文网址:英文网址:在95℃加热2分钟时,酶活性被完全激活。专利的酶/缓冲液配方,能耐受更多的循环数(45-50个循环),并且与需要较长预变性时间的热循环程序(95℃,10min)兼容。对GC含量较高(60%以上)的DNA片段或DNA模板中有PCR反应抑制剂存在时,此缓冲液配方也能保证扩增顺利进行。GoTaq®qPCRMasterMix系统提供最小的批与批之间的差异,在宽广的线性范围内保证高效、敏感的扩增。2.产品组分及储存条件ProductSize(50μl反应体系)Cat.#GoTaq®qPCRMasterMix200反应A6001包含:5×1mlGoTaq®qPCRMasterMix,2X100μl100XCXRReferenceDye2×13mlNuclease-FreeWaterProductSize(50μl反应体系)Cat.#GoTaq®qPCRMasterMix1000反应A6002包含:25×1mlGoTaq®qPCRMasterMix,2X5×100μl100XCXRReferenceDye10×13mlNuclease-FreeWater储存条件:GoTaq®qPCRMasterMix采用-20℃运输。到货后,将全部组分储存于-20℃,避光保存。使用过程中,所有组分可避光2-8℃保存3个月。普洛麦格(北京)生物技术有限公司Promega(Beijing)BiotechCo.,Ltd电话:8008108133,01058256268中文网址:英文网址:一般注意事项3.A光谱特性GoTaq®qPCRMasterMix采用的专利染料BRYTGreen®dye具有与SYBR®GreenI相似的光谱特性,激发波长:493nm,发射波长:530nm。CXR参比荧光染料具有与ROX™相同的光谱特性,激发波长:580nm,发射波长:602nm。采用GoTaq®qPCRMasterMix进行基因定量时使用与SYBR®GreenI和ROX完全相同的设置。3.BMgCl2浓度GoTaq®qPCRMasterMix中的MgCl2浓度已进行优化以达到最佳表现。如需对镁离子浓度进行调整,请加入PCR级的高浓度镁离子储存液(未提供)3.C兼容仪器GoTaq®qPCRMasterMix可用于任何能够检测SYBR®GreenI或FAM™染料的仪器。GoTaq®qPCRMasterMix中已含有低浓度的CXR参比染料。对于以下仪器,可直接使用,无需再加入参比荧光染料:AppliedBiosystems7500and7500FASTReal-TimePCRSystemBio-RadCFX96Real-TimePCRDetectionSystemBio-RadDNAEngineOpticon®andOpticon®2RealTimePC
本文标题:qPCR中文操作手册
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