Primer-BLAST step by step

About 7 min read · Updated 2026-09-30

Primer-BLAST is NCBI's free tool that designs primers while checking them against the whole genome or transcriptome for off-target amplification. You can also enter existing primers just to check specificity. The page looks long, but only a few fields usually need changing.

Try it with the tool

Design or check primers in the WetLab Kit primer tool, then press "Check with Primer-BLAST" to open NCBI with the primers and organism already filled in.

Open primer design & check →

1. PCR Template

Enter the gene's RefSeq accession (e.g. starting with NM_) if possible — only then can exon boundaries be used.
A pasted FASTA sequence works, but exon-junction features are limited.
To restrict primers to a region, fill in the Range From/To positions.
To only check existing primers, leave the template empty and enter the two primers under Primer Parameters.

2. Primer Parameters — size and Tm

Field	qPCR	Standard PCR
PCR product size	70 – 200	300 – 1000 (as needed)
Primer melting temperatures (Min/Opt/Max)	58 / 60 / 62	57 / 60 / 63
Max Tm difference	2–3	3

Entering primers in "Use my own forward/reverse primer" checks just those primers, or finds a partner if you enter only one.

3. Exon/intron selection — the key for qPCR

Exon junction span: choosing "Primer must span an exon-exon junction" places one primer across an exon junction. It cannot bind genomic DNA, so incomplete DNase treatment is less of a problem.
Intron inclusion: "Primer pair must be separated by at least one intron" puts the primers in different exons, so a genomic product would be much larger and distinguishable.

Both require a RefSeq accession as the template. For single-exon genes, rely on a no-RT control instead.

A primer spanning an exon–exon junction binds cDNA made from mRNA, but on genomic DNA its two halves are separated by an intron, so it cannot bind.

4. Specificity checking — what to compare against

Database: "Refseq mRNA" for qPCR on cDNA; "Refseq representative genomes" or "Genomes for selected organisms" for genomic PCR.
Organism: always enter the species you work with (e.g. Homo sapiens, Mus musculus). Leaving it empty compares against everything — slow and cluttered.
Leave the other specificity settings (allowed mismatches, etc.) at their defaults.

Finally press Get Primers; results appear after a minute or so.

5. Reading the results

Each primer pair lists sequence, Tm, GC and self-complementarity, plus "Products on target templates" (products from your gene).
"Products on potentially unintended templates" means other genes may also amplify. It is riskier when the product size is similar and the mismatches are not near the 3′ end.
Products only on several transcript variants of the same gene are usually fine — often you want primers that measure all variants.
Lower self-complementarity and self 3′-complementarity scores are better.

Results split into products on the intended gene (top) and unintended products (below). Unintended products are riskier when similar in size and without mismatches near the 3′ end.

6. Common mistakes

No organism entered, so the results never end
qPCR with the default product size (up to 1000 bp)
Entering the reverse primer in template orientation — both must be written 5′→3′
Trusting Primer-BLAST alone — efficiency, melt curve and product size on a gel still need checking at the bench