Primer design · check

Paste a sequence → suggested primer pairs · Tm, GC, dimer and hairpin checks · specificity via NCBI Primer-BLAST
Free · runs entirely in your browser

1. Template sequence

Paste the mRNA (cDNA) sequence of the gene to amplify. FASTA (a line starting with >) and GenBank sequences work as-is. You can copy sequences from NCBI Gene/Nucleotide as "FASTA".

2. Purpose

Detailed settings (usually leave as-is)
Product size – bp · target Tm °C (± )
Region to include – (e.g. a knockdown site or mutation. Leave empty for anywhere)

Primer sequences

Add a template (optional — calculates product size and position)
Reaction conditions (used for Tm — usually leave as-is)
Na⁺/K⁺ mM · Mg²⁺ mM · dNTP mM · primer nM
Defaults match the widely used Primer3 conditions. qPCR master mixes contain about 3 mM Mg²⁺, so the real Tm is 1–3 °C higher. Vendor calculators differ by a few degrees — that is normal.

Check specificity with BLAST

This tool checks the primers themselves (Tm, GC, dimers). Whether they also bind other genes (specificity) requires a comparison against the whole genome, done in NCBI Primer-BLAST. The "Check with Primer-BLAST" button opens NCBI in a new window with the primers filled in — sequences are sent to NCBI only then, and this site sends nothing. See the Primer-BLAST how-to guide for an illustrated walkthrough.
Organism