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Optimizing PCR Primer Design: Tips & Tools for Efficient Amplification Detection

Learn how to design primers for PCR, avoid common pitfalls, and use analysis tools for precise results. Explore real-time vs. traditional PCR and post-processing techniques.

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Optimizing PCR Primer Design: Tips & Tools for Efficient Amplification Detection

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  1. General guidelines for primer design • 18-30 nucleotides • G/C content: 40-60% • Avoid complementary sequences of primers (especially at the 3’ end) • Avoid mismatches at the 3’ end • Avoid 3 or more G or C at the 3’ end • Avoid a 3’ end T

  2. Primer design to eliminate amplification from contaminating genomic DNA

  3. Primer design to detect amplification from contaminating genomic DNA

  4. Primer design and analysis tools • Vector NTI • Primer 3 • IDT SciTools PrimerQuest Oligo Analyzer

  5. Real-time PCR • QPCR • Real-Time Vs Traditional PCR Post PCR processing Poor agrose gel resolution (semi-quantitative) Detect end point of the reaction

  6. http://www.appliedbiosystems.com/support/tutorials/pdf/rtpcr_vs_tradpcr.pdfhttp://www.appliedbiosystems.com/support/tutorials/pdf/rtpcr_vs_tradpcr.pdf

  7. http://www.appliedbiosystems.com/support/tutorials/pdf/rtpcr_vs_tradpcr.pdfhttp://www.appliedbiosystems.com/support/tutorials/pdf/rtpcr_vs_tradpcr.pdf

  8. http://www.appliedbiosystems.com/support/tutorials/pdf/rtpcr_vs_tradpcr.pdfhttp://www.appliedbiosystems.com/support/tutorials/pdf/rtpcr_vs_tradpcr.pdf

  9. ΔCt, ΔΔCt method to detect fold change

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