How it works & how to read the results ▼
Pipeline: your gene → its RefSeq mRNA (via mygene.info) → sequence + exon map from NCBI → Primer3 designs candidate pairs over the transcript. Nothing is pre-canned — primers are generated fresh for the current reference transcript.
Specific isoform? Instead of a gene name you can enter an exact transcript — a RefSeq accession (NM_000546) or an Ensembl id (ENST… human, ENSMUST… mouse) — and primers are designed on that transcript. Species is auto-detected for transcript IDs.
Exon-junction badge: EXON-JUNCTION = a primer sits directly on an exon–exon boundary (gold standard — it cannot bind genomic DNA). SPANS INTRON = the amplicon crosses a junction, so genomic DNA would give a larger product. Pairs with these properties are ranked first.
Defaults: Tm ≈ 60 °C (±2), length 18–25 nt, GC 40–60 %, GC-clamp, amplicon 70–200 bp — standard SYBR-Green qPCR settings.
Always verify: hit UCSC In-Silico PCR ↗ on any pair to confirm it amplifies a single, correct product against the genome before you order. Then grab the order CSV for IDT / Sigma bulk entry.