Individual skill
primer-design
Designs and audits PCR and RT-qPCR primers with Primer3, explicit thermodynamic conditions, reference-based off-target amplification searches, and traceable sequence coordinates. Use for designing primer pairs, checking existing primers, exon-junction or isoform-specific assays, variant masking, cloning tails, multiplex compatibility, and interpreting Primer-BLAST results. Includes bounded local in-silico PCR and BLAST screening; distinguishes computational candidates from experimentally validated assays.
skills/primer-design/SKILL.md
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# Primer design and specificity
Produce candidate oligos in 5-prime-to-3-prime orientation, with the exact template,
chemistry, intended products, and search scope behind each conclusion. Calculate
sequence-dependent quantities with the supplied tools. A familiar gene name, good
Primer3 penalty, or a single BLAST alignment cannot establish primer specificity.
## Choose the workflow
| Request | Start here |
| --- | --- |
| New genomic PCR or RT-qPCR pair | Define the assay and reference; design; assess thermodynamics; screen products. |
| Check an existing pair | Prepare pair TSV; assess both full oligos and annealing cores; screen with explicit intended coordinates. |
| Exon junction, transcript isoform, allele discrimination | Read [design-workflows.md](references/design-workflows.md); supply sequence annotation before imposing constraints. |