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<p><span class="autocomment">Manual and Protocols</span></p> <p><b>New page</b></p><div>{{BioMicroCenter}}<br /> <br /> == Training Session Notes: ==<br /> * Highly multiplex PCR.<br /> <br /> === Assays ===<br /> TaqMan<br /> Intercalating Dye (EvoGreen)<br /> Roche UPL<br /> <br /> Genotype w/ Taqman - recommend 50 copies per cell. 50ng. 30ng/ul starting material<br /> <br /> miRNA<br /> <br /> Single Cell (requires Taqman)<br /> <br /> === WorkFlow ===<br /> * Input<br /> 1ng - 250ng is standard input<br /> Cell direct Kit from Invitrogen for single cells <br /> Use 2-3 lines per sample as replicates.<br /> <br /> * Specific target preamplification<br /> <br /> 48/96 Asssys mix (@180nM)<br /> cDNA sample<br /> preamp master mix<br /> <br /> <br /> 14 (reg) / 18 (1 cell) cycles of PCR<br /> <br /> Dilute 1:5<br /> <br /> * prep assays + samples<br /> <br /> 2x Master mix 2.50ul / sample - NEEDS ROX - ABI universal master mix<br /> Loading reagent 0.25ul / sample (detergent to reduce surface tension)<br /> cDNA 2.25ul pre amp<br /> <br /> 20x primer probe 2.5ul (TaqMan)<br /> Loading Reagent2 2.5ul detergent<br /> <br /> <br /> * prime chip<br /> * pipette samples + assays<br /> * load / mix chip<br /> * RT-PCR<br /> * Data Analysis<br /> <br /> Plots are heatmaps and RT curves<br /> similar plots for deltaCT and DDCT<br /> <br /> === Chip Types ===<br /> * Dynamic PCR: 48x48 or 96x96 - relative quantification<br /> * Digital PCR: 12x765 or 48x770 - rigorous quantification<br /> <br /> Big raised areas on array are the Air Tanks<br /> <br /> 48.48 - 9nL of sample 1nL of assay<br /> 96.96 - 6.7nL of sample 0.7nL of assay<br /> mix by diffusion<br /> <br /> === Manual and Protocols===<br /> Manual and Protocol from Fluidigm are available.<br /> <br /> * [[Media:Fluidigm_RT-_PCR_Analysis_v2.1_User_Guide_.pdf|Fluidigm RT- PCR Analysis Softwarev2.1 User Guide]] <br /> * [[Media:Fluidigm_RT_PCR_Analysis_brief_manual_07302010.doc|Fluidigm_RT_PCR Analysis brief Protocol]] <br /> * [[Media:Fluidigm_96.96_RT-_PCR_Quick_Reference_.pdf | Fluidigm 96.96 RT- PCR Quick Reference ]]</div>

imported>Manlin Luo