PCR Primer Designer — Design Primer Pairs Online
Design ranked PCR primer pairs from a template, with Tm, GC and dimer checks.
Design PCR primers from a DNA template online. Paste a sequence, optionally marking a region the product must span, set your length, Tm, GC and amplicon-size preferences, and get ranked forward/reverse primer pairs scored on nearest-neighbor Tm, GC content, 3′ clamp, hairpins and cross-dimers. Copy the primers or export the whole set as CSV. The qPCR section below designs the same template as a real-time assay instead: a 70–150 bp product, primers matched within 2 °C, and a hydrolysis probe between them that never begins with G.
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Candidate primers are scored on length, nearest-neighbor Tm, GC content, 3′ GC clamp and self-structure, then paired by amplicon size, Tm match and cross-dimer. Tm uses SantaLucia (1998) thermodynamics under the conditions set above. Verify specificity against your template or genome before ordering.
qPCR primers and a TaqMan probe
Designs a qPCR primer pair and a hydrolysis (TaqMan) probe from the template above. The amplicon is 70–150 bp, short enough to double every cycle (the default search above extends to 1,000 bp). The two primer Tm values match within about 2 °C, and the probe sits between them, 6–12 °C warmer than the primers so that it is bound before they extend.
Paste a template above to design an assay from it.
Design, edit, and verify complete constructsSeqStudio combines sequence editing and annotation, plasmid maps, primer/cloning/CRISPR design, Sanger verification, and GenBank/SnapGene files in one workspace.
How to use the Primer Designer tool
- 1Paste a DNA template (raw or FASTA), and optionally mark a target region.
- 2Adjust the length, Tm, GC and amplicon-size constraints, or keep the defaults.
- 3Click Design primers, then copy the best pair or export all candidates as CSV.
- 4For a real-time assay, use the qPCR section below: it designs primers and a TaqMan-style probe from the same template.
Frequently asked questions
How are primers scored and ranked?
Each candidate is scored on how close its length, nearest-neighbor Tm and GC content are to your targets, plus a 3′ GC-clamp check and self-dimer/hairpin screening. Pairs are then ranked by combined penalty, the Tm match between the two primers, and cross-dimer ΔG.
Does it check primer specificity against my genome?
No. It designs primers from the template you paste. To confirm a pair amplifies only your intended target, check them with the In-silico PCR tool against your full template, or use a specificity search before ordering.
What melting-temperature model does it use?
SantaLucia (1998) nearest-neighbor thermodynamics under the reaction conditions you set (oligo, Na⁺, Mg²⁺, dNTP), the same engine as the Oligo Analyzer.