Use this free online PCR primer design tool to choose forward and reverse primers from a DNA sequence using routine settings, or add cloning overhangs and restriction sites when needed.
Useful Checks
The current sequence can also be opened in Restriction Site Analyzer, which connects primer design with cloning-site checks.
After generating primers, open the primer pair directly in Primer Binding Checker to check binding positions and expected amplicon size on the same template.
Use fixed primer length when you want simple primers of a set size, or use target Tm when matching primer melting temperature matters more.
In target Tm mode, forward and reverse primers can be different lengths because each binding region is adjusted separately toward the same Tm.
Restriction-site overhangs use built-in enzyme-specific flanking-base rules, while binding-region Tm stays focused on the bases that bind the template.
Minimum Inputs Needed
DNA sequence. The default primer length and calculation settings can be used for a routine first pass.
Optional Settings
Template topology
Use linear for normal DNA fragments. Use circular / plasmid for plasmids or when the target may cross the coordinate origin, meaning base 1 / the start of the entered sequence, not the biological ori.
Primer length
Sets a fixed binding-region length when Target Tm is blank. The default is suitable for a routine first pass.
Amplicon region (optional)
Enter the part of the template you want to amplify. Leave it blank to use the full sequence.
Target Tm, deg C (optional)
Use this if you want primers near a specific melting temperature. Leave blank if primer length matters more.
Forward / reverse custom 5' overhang
Add extra bases to the 5' end of a primer. These bases do not bind the template in the first PCR cycles.
5' flanking bases before restriction site
Extra bases before a restriction site can help digestion. Auto uses built-in enzyme-specific flanking-base rules and is suitable for most cloning designs.
Forward / reverse restriction site
Add an enzyme site to the primer. Search by enzyme name or type the site sequence.
Na+, Mg2+, primer, and dNTP settings
These change the Tm estimate. Keep defaults unless you know your PCR conditions.
How To Use
Paste or upload the DNA sequence.
Choose linear or circular topology.
Enter an amplicon region only if you want a specific part of the sequence.
Change primer length or target Tm only if needed.
Add overhangs or restriction sites only for cloning designs.
Click Design primers.
Understanding The Results
The main outputs are the forward and reverse primers, written 5' to 3'.
The binding-region Tm, GC%, length, homopolymer, and 3' end checks help you judge the design.
Hairpin, self-dimer, 3' self-dimer, cross-dimer, estimated structure Tm, and approximate Delta G results provide browser-side structure screening.
Overhangs and restriction sites are included in the primer sequence, but not in binding-region Tm.
Copy either primer, transfer the generated pair to Primer Binding Checker, or open the external Tm calculators for an additional protocol-specific check.
Accepted Input Formats
Raw DNA sequence.
FASTA. Header lines are ignored.
GenBank text with an ORIGIN sequence section. Feature annotations, including ori/origin-of-replication annotations, are ignored.
Multiple FASTA records are concatenated into one template. Analyze one record at a time to avoid artificial junctions.
.dna / SnapGene binary files are not supported. Export as FASTA or GenBank.
Assumptions And Limitations
This is a primer planning tool, not a PCR guarantee.
Tm is an estimate and depends on the salt and primer settings.
Hairpin and dimer values are screening estimates, not full folding predictions.
Long overhangs, restriction sites, and difficult templates may still need manual checking.
The browser tool accepts sequence files smaller than 2 MB and templates up to 500,000 bp.
Always verify final primer sequences before ordering.
Example
Paste your insert sequence, keep the default primer length, add EcoRI to the forward primer and HindIII to the reverse primer if needed, then design and check the primers.
Use note: These tools are for research and educational planning. Check important calculations and sequence designs before ordering reagents or running experiments.