Primer Design

Primers can be designed directly against the sequences annotated in CnidoSite, or against a sequence you paste in. Design is performed by primer3 (version 2.6.1) running on this server — your sequence is not sent to any third-party website. For reference, the primers released with this database were designed with an optimal melting temperature of 60°C (58–62 °C), an optimal primer length of 20 bp (18–24 bp), and a GC content between 40% and 60%, with self-complementarity restricted to limit primer-dimer formation; that parameter set is the CnidoSite published protocol preset below.

Template taken from CnidoSite: PcrG010845.mRNA1 (PcrG010845.mRNA1), 981 bp.
Template

Two ways to supply a template: pick a species and a gene / transcript ID, or paste your own sequence. If the gene ID is found it is used; the pasted sequence is used otherwise. A template must be at least 30 bp.

Load example sequence
Primer parameters
Advanced parameters (any field left empty keeps the preset value)

A value that is not a number is ignored. The product size range is applied only when both of its fields are filled in and the maximum is at least the minimum.

Melting temperature
Primer length and GC content
Product and output
Self-complementarity and probe
Download TSV
Design results sorted by primer3 penalty, lowest first

Template 981 bp · parameter preset CnidoSite published protocol (Tm 60 [58-62] °C, 20 [18-24] bp, GC 40-60%) · 5 primer pairs returned.

Pair Left primer (5'→3') Position Len Tm (°C) GC (%) Right primer (5'→3') Position Len Tm (°C) GC (%) Product (bp) Penalty
1 GACCACATGGGAGAAGTGCA 646–665 20 59.963 55.000 CAACGGCCAGGACTTTAGGT 738–757 20 59.964 55.000 112 0.073
2 AGGACCAGCATGCTTCCTTC 317–336 20 60.035 55.000 CTTGATTGCTGCAGGATGGC 473–492 20 59.898 55.000 176 0.137
3 GAGGACCAGCATGCTTCCTT 316–335 20 60.035 55.000 CTTGATTGCTGCAGGATGGC 473–492 20 59.898 55.000 177 0.137
4 GCCATCCTGCAGCAATCAAG 473–492 20 59.898 55.000 CAACGGCCAGGACTTTAGGT 738–757 20 59.964 55.000 285 0.139
5 GCCATCCTGCAGCAATCAAG 473–492 20 59.898 55.000 TGCACTTCTCCCATGTGGTC 646–665 20 59.963 55.000 193 0.139
Primer map pair 1 (lowest penalty) · product 112 bp at template position 646–757 · the two tracks are drawn at different scales

Left primer — binds the template strand as listed (5′→3′)Right primer — its reverse complement binds hereProduct

Template981 bpproduct 112 bp · template 646–757product 112 bpleft primer, template 646–665right primer anneals here, template 738–7571246491736981Amplicon112 bpleft primer GACCACATGGGAGAAGTGCA · 5′→3′right primer CAACGGCCAGGACTTTAGGT · its reverse complement binds the template strandGACCACATGGGAGAAGTGCACAACGGCCAGGACTTTAGGTinterior 72 bp646674702729757
Left primer
GACCACATGGGAGAAGTGCA
template 646–665 · 20 bp · Tm 59.963 °C · GC 55.000 %
Right primer
CAACGGCCAGGACTTTAGGT
template 738–757 · 20 bp · Tm 59.964 °C · GC 55.000 %

Amplicon sequence (5′→3′ on the template strand, 112 bp) — blue is the left primer, red is where the right primer anneals. Copy from here to order or to check a base by eye.

GACCACATGGGAGAAGTGCAATGTGAAGTGTTCAGTGAACCAACAAATGAGTGGCAGTTTATTTCGATGTTTGGCATAAATCCCAGCTTATTACCTAAAGTCCTGGCCGTTG

The right primer is listed as its own 5'→3' sequence; what anneals to the template strand shown here is its reverse complement, so the highlighted stretch is complementary to the listed sequence. That is expected.

How to read this table. Position is a 1-based interval on the template, and both primers use the same convention: it is the stretch the primer occupies on the template strand (for the left primer, starting at its 5’ end; for the right primer, the region it anneals to, i.e. where the reverse complement of the listed sequence sits on the template). The two coordinates in a row can therefore be subtracted directly, and the amplicon view above marks both of them so you can check them by eye. Penalty is primer3’s weighted sum of the deviations from your constraints — lower is better, and the pairs are returned in that order. Tm is computed by primer3 from the SantaLucia thermodynamic parameters at the salt and primer concentrations in effect. The primer sequences themselves are 5’→3′ and can be ordered as they stand; the right primer anneals to the template as its reverse complement.

Specificity has not been assessed. This page only designs primers; it does not check whether they also anneal elsewhere in the genome or transcriptome. Once you have candidates, confirm each primer sequence with this site’s BLAST. The Tm and GC settings above are the design targets, and the annealing temperature still has to be optimised at the bench.

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