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About PCR Primer Design: From Melting Temperature to Validated Assays

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You will learn to design oligonucleotide primers that amplify one intended locus and nothing else. Starting from base pairing and duplex thermodynamics, you will predict melting temperature with the nearest-neighbor model, balance GC content, length and 3' anchoring, and eliminate hairpins, self-dimers and primer-dimers before ordering. You will then check specificity computationally against a reference genome, place amplicons deliberately (including across exon junctions for RT-qPCR), validate amplification efficiency with a standard curve, and append tails and adapters for cloning and sequencing workflows.

Estimated Time to Complete

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What You'll Learn

Concepts:
GC Clamp Primer Length Selection Amplicon Length and Placement Degenerate Primer Design Primer Pair Experimental Validation Exon-Junction-Spanning Primers Primer GC Content PCR Amplification Efficiency Primer-Dimer Formation Annealing Temperature Optimization Primer 3' End Specificity In-Silico PCR Specificity Checking Wallace Rule for Melting Temperature Primer Tails and Adapter Sequences PCR Primer Function Primer Pair Tm Matching Primer Hairpin Structure Nearest-Neighbor Melting Temperature Calculation

What you will learn

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