1、本文档共22页，可阅读全部内容。 2、本文档内容版权归属内容提供方，所产生的收益全部归内容提供方所有。如果您对本文有版权争议，可选择认领，认领后既往收益都归您。 3、本文档由用户上传，本站不保证质量和数量令人满意，可能有诸多瑕疵，付费之前，请仔细先通过免费阅读内容等途径辨别内容交易风险。如存在严重挂羊头卖狗肉之情形，可联系本站下载客服投诉处理。 文档侵权举报电话：400-050-0739(电话支持时间：9:00-19:00)。 Maximizing the Efficacy of Melt Profiling through Stringent PCR Optimization Steven F. Dobrowolski, PhD Adapting a Customer’s PCR Assay to DB/HRTD Optimizing PCR to a Capillary System and Initial Profiling Steven F. Dobrowolski, PhD First QuestionIs the customer currently using the LightCycler? If yes, the job of adapting a PCR assay for melting analysis is much simpler. Real-time PCR is Extremely Useful to Assess Amplification Adapting a LightCycler Assay Reaction volume of 10μl Hot-start polymerase, Klen Taq in a complex with TaqStart antibody Add LCGreen to PCR mastermix (1μl/reaction) and omit an equal amount of water. Single fluorescence acquisition in Channel 1 at end of extension phase. Use their established amplification parameters but add initial denaturation and re-annealing at end Modifying a Typical PCR Cocktail10 reactions, 10μl volumes Customer PCR FRET Assay 10X buffer 12 10X dNTP 12 Fwd Primer 6 Rev Primer 6 Probe 1 6 Probe 2 6 Enzyme 2.4 H20 57.6 Add 9ul + 1ul DNA PCR with LCGreen 10X buffer 12 10X dNTP 12 10X LCGreen 12 Fwd Primer 6* Rev Primer 6* Taq/antibody 12 H20 48 Add 9ul + 1 DNA Potentially Successful Try Something New Customer’s Assay’s are Developed for Block-Style Cycler This is harder, but by no means impossible Ask for a PCR assay that is robust, free of undesired product, and between 100-250 bp Ask to see a gel of the chosen product. Avoid a fragment that requires DMSO, Glycerol or other adjuvant in the PCR. Chemistry and Cycling will require modifications Chemistry Optimization for PCR in Capillaries- where to begin Use a 10μl reaction volume Use Idaho Technology buffers, start with 20mM MgCl2 Use hot-start with Klen taq in a complex with TaqStart antibody Use recommended primer concentration Use 15-30ng template DNA Cycling ConditionsBlock to CapillaryMajor changes required for Adaptation to PCR in a Capillary System Cycling Protocol has 3 Components Initial Denaturation (high temperature step) 1-2 minut

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