qPCR Primer designer
This is a simplified, easy-to-use version of the NCBI Primer-BLAST application with a smaller subset of the important options. Submitting runs NCBI Primer-BLAST via our server proxy; results appear below once NCBI finishes (often 30–90 seconds).
WHAT IT’S FOR
You need qPCR primers that amplify the right product from your template and don’t hit the wrong transcripts in your organism. NCBI Primer-BLAST designs primers with Primer3 and then BLASTs them against RefSeq mRNA to filter off-target pairs — but the full web form is long and easy to misconfigure.
This page is the “design me a sensible qPCR pair” step: look up a standard
NM_ RefSeq mRNA from a gene name, set product size and melting temperature, pick an
unambiguous organism from NCBI Taxonomy, and read off primer sequences plus a template map showing where
each pair binds. See the
primer design guide for the wider workflow.
It is not a replacement for checking amplicon splicing, SNPs, or lab validation. Treat output as a strong starting point, then verify in silico and at the bench.
HOW IT WORKS
1. Template
Enter a gene symbol and organism to fetch a RefSeq mRNA accession (NM_…), or paste an
accession or raw sequence into the PCR template box. Accessions are preferred — NCBI handles exons
correctly and the template map can show 5′ UTR, exons, and 3′ UTR from GenBank.
2. Organism
Autocomplete queries NCBI Taxonomy so Primer-BLAST gets one exact scientific name (e.g.
Rattus norvegicus, not “rat”). That organism limits the specificity database
search.
3. Primer design on NCBI
Your settings are posted to NCBI Primer-BLAST through our PHP proxy (there is no official API). If your template matches other RefSeq mRNAs (common for genes with many isoforms, e.g. TP53), NCBI may pause for a transcript checklist — we automatically select All similar targets and continue. Hidden defaults include RefSeq mRNA specificity checking, automatic search mode, and up to ten primer pairs returned. Primer GC content is set to 35–65% by default; if no primers are found, the tool automatically retries with 20–80% GC.
4. Results
We obtain primer sequences, Tm, GC%, product length, and binding coordinates from the finished job. Near-duplicate pairs (similar forward start and product size) are collapsed to keep the list readable. For RefSeq templates, an SVG map shows primer binding sites on the mRNA.
HOW TO USE IT
-
Enter a gene (e.g.
GAPDH) and organism, then click Look up NM accession — or pasteNM_…/ sequence directly into PCR template. - Pick an organism from the NCBI taxonomy suggestions (type at least two letters).
- Set PCR product size (min/max slider), optimal primer size, Tm, and specificity stringency (each sent as opt ± 2 to NCBI for size/Tm).
- Click Design primers and wait — jobs often take 30–90 seconds.
- Read the primer pairs table; click a row or pair button to update the template map below.
READING THE RESULTS
- Forward / reverse sequences — reported 5′ → 3′; check length, Tm, and GC% against your lab preferences.
- Product (bp) — expected amplicon on the template you submitted; confirm it spans the region you intend (e.g. one exon for RNA, or known splice form).
- Template map — forward (teal) and reverse (orange) binding on the plus strand; yellow band = PCR product. Exon/UTR layout applies to RefSeq accessions only.
If no primers return, try widening product size, checking the template accession, or confirming the organism matches your sample. The status line notes when GC limits were relaxed automatically.