biorad定量pcr說明書-資料下載頁
2025-05-14 09:01本頁面
【正文】 tate Ct : Thymus Ct : ? Only possible with DNA Binding dyes (SYBR Green I) and after pleted real time PCR ? Determines the temperature at which 50% of the DNA molecules separate into two strands or “melts” apart ?Discriminates by Melting Temperature (Tm), Tm is dependent on: sequence (G/C content) length What is Melt Curve Analysis? Fluorescence vs. Temperature change in fluorescence single amplified product Tm Melt curve showing two amplified products Two amplified products Tm Tm Melt Curve Check specificity of the reaction Collecting at Higher Temperatures。 does that work to avoid detection of primer dimers? Collecting at 82 186。C would record specific product only Primer Dimer’s Impact on the Assay Outline ? Real Time PCR and Conventional PCR ? Introduction to Quantitative PCR ? Ct value and RealTime Quantitative PCR Theory ? Laboratory Technique ? Primer Design for RealTime PCR Laboratory Technique Good Laboratory Technique ? Do not underestimate the importance of using: Screw cap tubes Aerosol barrier tips Hotstart polymerase Master mixes Replicates Serial dilutions ? And the golden rule: – Pipet only once into each well Same Reagents, Different Hands Good Technique Poor Technique Cycle Cycle Outline ? Real Time PCR and Conventional PCR ? Introduction to Quantitative PCR ? Ct value and RealTime Quantitative PCR Theory ? Laboratory Technique ? Primer Design for RealTime PCR Primer Design for RealTime PCR Primer Design for RealTime PCR Targets an amplicon length of 75 to 200 bp 50 to 60% overall GC content Limit secondary structure Limit stretch of G or C’s longer than 3 bases No stable interactions between primers (primer/dimer) Place C’s and G’s on ends of primers, but no more than 2 in the last 5 bases on 3’ end Primer Design: Step by Step 1. Evaluate target sequence for secondary structure. 2. Identify regions of ~100 bp as potential amplicons. 3. Scan sequence by eye or use a program to select potential primer pairs.* 4. Use oligo analysis tools to check primers for: Dimerization Secondary Structure Cross Reactivity *Repeat if necessary 謝 謝 ！
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