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.

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Presentation transcript:

General guidelines for primer design 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

Primer design to eliminate amplification from contaminating genomic DNA

Primer design to detect amplification from contaminating genomic DNA

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

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

ΔCt, ΔΔCt method to detect fold change