Primer Design Tool Help

Design forward and reverse PCR primers for a fixed binding length or target Tm. Add restriction sites and custom 5' overhangs for cloning, then check the resulting pair against your template.

After designing, use Download format to save both primers as FASTA or a TSV table. Copy buttons and transfers remain available.

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.
Send results to Primer Scan (Beta) to check the full generated pair against a host genome or custom reference. The input template is not transferred.
Leave Target Tm blank to use a fixed binding length, 25 bases by default. Enter a target Tm to adjust each primer's binding length separately. Added 5' tails do not count toward binding length or binding-region Tm. Target Tm is a melting-temperature target, not the PCR annealing temperature.
Restriction-site overhangs use built-in enzyme-specific flanking-base rules, while binding-region Tm stays focused on the bases that bind the template.
If the forward and reverse restriction enzymes have different recognition sites but leave compatible cohesive ends, the tool warns that the resulting fragments can still ligate.

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 25 bases; added 5' tails are not included.
Amplicon region (optional): Enter a 1-based, inclusive range such as 100-900. Leave blank for the full template. In circular mode, a start greater than the end, such as 900-100, selects a region crossing base 1.
Target Tm, deg C (optional): Enter a target melting temperature between 30 and 95 degrees C. This overrides fixed primer length; each binding region is adjusted separately. Target Tm is not the PCR annealing temperature.
Forward / reverse custom 5' overhang: Add bases before the binding region. If a restriction site is also selected, the order is custom overhang, flanking bases, restriction site, then binding sequence. The added tail is excluded from binding-region Tm.
5' flanking bases before restriction site: Auto adds the stored enzyme-specific 5' clamp, with GCGC as the fallback when no rule is available. Choose None or a listed sequence to override it. Confirm the required flanking bases with the enzyme supplier.
Forward / reverse restriction site: Add one restriction site per primer. Search by enzyme name or type one DNA/IUPAC motif. A custom motif can contain ambiguity codes; the template must contain A, C, G and T.
Na+, Mg2+, primer, and dNTP settings: Enter Na+ equivalent, Mg2+ and total dNTP in mM, and primer concentration in nM. Total dNTP is the sum of all four nucleotides: 0.2 mM each means 0.8 mM total. Use reaction concentrations, not stock concentrations.

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.
Changing design inputs clears the previous result. Click Design primers again after correcting inputs.

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.
A compatible cohesive-end warning means the selected enzymes can create ligatable ends even though their recognition sites differ. The ligation junction may not recreate either original site, so verify the planned orientation and junction sequence.
Copy either primer, transfer the generated pair to Primer Binding Checker, or open the external Tm calculators for an additional protocol-specific check.

Open Show detailed primer metrics to inspect the binding-region and pair-compatibility tables. On narrow screens, scroll each table horizontally to see all columns. Use Tab to focus an overflowing table, then use the Left and Right arrow keys to scroll.

Accepted Input Formats

Raw A/C/G/T DNA sequence. Ambiguity codes are accepted in custom restriction motifs, not in the template.
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

The tool designs primers against your supplied sequence. It does not screen genome databases for off-target amplification.
Tm uses SantaLucia nearest-neighbor parameters and an Owczarzy salt correction. Use your polymerase supplier's guidance to choose an annealing temperature.
Hairpin and dimer values are screening estimates, not full folding predictions.
Long overhangs, restriction sites, and difficult templates may still need manual checking.
Compatible-end warnings cover supported enzymes with fixed, unambiguous cohesive ends. Variable Type IIS or degenerate-site ends need the complete product sequence: check them in Restriction Site Analyzer and confirm cleavage patterns and ligation junctions with supplier documentation.
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

For a fixed-size design, paste your insert and leave Target Tm blank to use the default 25-base binding regions. To adjust both binding regions toward a melting-temperature target instead, enter Target Tm. Add restriction sites only when your cloning method needs them, then review the full primers and check their binding.

Cloning With A C-Terminal Tag

If translation must continue into a downstream tag, omit the insert's stop codon and keep the insert, cloning junction and tag in frame. Check that a stop follows the tag in the final construct. To omit the tag, place an in-frame stop before it.

Translate the assembled coding sequence, including the junction and tag, with ORF and Protein Translator. Check the expected amino-acid sequence; Primer Designer does not automatically validate fusion reading frames. See Thermo Fisher's PCR cloning considerations and your vector documentation.

Use this result with other tools

Before design: Open in Restriction Site Analyzer sends the validated template and topology to Restriction Site Analyzer for site mapping. Open in Primer Binding Checker beside the input sends the same template to Primer Binding Checker; enter the primers you want to test there.
After design: Open generated primers in Primer Binding Checker in the results also sends the generated forward/reverse pair and selected amplicon region. Run Check primer binding there to compare the predicted product with your intended region, including any circular origin crossing.
Send results to Primer Scan: sends the full named primer pair to Primer Scan. Select the host or custom reference and run the scan there. The template is not transferred by this button.

A cloning tail can be absent from the original template. Review the receiving tool’s 5′-overhang and binding settings when checking a tailed primer. Transfer buttons do not transfer every reaction setting; confirm the receiving settings before running.

Correcting highlighted input

When the main action rejects pasted sequence text, unsupported characters are highlighted in the field and the error message explains the problem. Correct the marked characters using the alphabet allowed by this tool, then run it again. The highlights update as you edit. A malformed FASTA or GenBank record may require a structural correction even when no individual character is marked.

If the tool does not load

The loading message remains visible while the required scripts arrive. If a script cannot load, the tool shows a connection message and keeps its main actions disabled. Copy any sequence inputs before choosing Reload tool. Check your connection and retry; copied inputs can be restored after the reload.

References

Primer design methods and data sources.

Use note: These tools are for research and educational planning. Check important calculations and sequence designs before ordering reagents or running experiments.