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: gfas1.m1.15597.m1 (gfas1.m1.15597.m1), 2,810 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 2,810 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 AGCCAAGGGAAACTCTCTGC 2423–2442 20 59.963 55.000 GTTACAACACGGTTGCCACC 2686–2705 20 59.970 55.000 283 0.067
2 CCAGGACAACACGGAGGTAC 131–150 20 60.038 60.000 TCGGCGTCTGATCATCGTTT 260–279 20 59.827 50.000 149 0.210
3 CCAGGACAACACGGAGGTAC 131–150 20 60.038 60.000 TTCGGCGTCTGATCATCGTT 261–280 20 59.827 50.000 150 0.210
4 CCAGGACAACACGGAGGTAC 131–150 20 60.038 60.000 TTTCGGCGTCTGATCATCGT 262–281 20 59.827 50.000 151 0.210
5 GAAAGTGATGGGCCAGGACA 119–138 20 59.962 55.000 TCGGCGTCTGATCATCGTTT 260–279 20 59.827 50.000 161 0.211
Primer map pair 1 (lowest penalty) · product 283 bp at template position 2,423–2,705 · the template track shows a 1,500 bp window of the 2,810 bp template — the blue segment on the top bar is where that window sits, and every scale is in template coordinates · the two tracks are drawn at different scales

Left primer — binds the template strand as listed (5′→3′)Right primer — its reverse complement binds hereProductWindow drawn in the template track

whole templatethe 1,500 bp drawn in the track below · template 1,311–2,810 of 2,810Template1,500 bp windowproduct 283 bp · template 2,423–2,705product 283 bpleft primer, template 2,423–2,442right primer anneals here, template 2,686–2,7051,3111,6862,0612,4352,810Amplicon283 bpleft primer AGCCAAGGGAAACTCTCTGC · 5′→3′right primer GTTACAACACGGTTGCCACC · its reverse complement binds the template strandinterior 243 bp2,4232,4942,5642,6352,705
Left primer
AGCCAAGGGAAACTCTCTGC
template 2,423–2,442 · 20 bp · Tm 59.963 °C · GC 55.000 %
Right primer
GTTACAACACGGTTGCCACC
template 2,686–2,705 · 20 bp · Tm 59.970 °C · GC 55.000 %

Amplicon sequence (5′→3′ on the template strand, 283 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.

AGCCAAGGGAAACTCTCTGCCTGTTTGCTCTCTCAACTCCTGCTAAGCCAAAGAAAAGGGACTGCCTGCAGTCTAGTGAAGTCAGTTAAGCTTGTAATGTCCTAATGTATAAACTGGTTCTTGTAACATGTGAAACGTGATTTGAGCAACAATGCCTGAGCTGGAAGTAGATAGAAGAGAGAAACTTTCTGATGACTGTTCCAAGCATACAGGGGTAGGTGCATGTTAAAAAACAAAAACATTTTCACTGTAACTTCCACCTTGGTGGCAACCGTGTTGTAAC

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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