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← Primer database

Primer database

COX5A

cytochrome c oxidase subunit 5A

Gallus gallus

PublicationXu et al. 2022

Sequences

Forward (5′→3′)

ACGCCCGCTGCTACTCCCA

Reverse (5′→3′)

CGCTGGCAAAGTCATTTAACCGT

Amplicon

204 bp

estimated from mapping

Primer Tm (°C)

ConditionsForwardReverseAverageΔ F/R
qPCR master mix70.665.368.05.3
Primer-BLAST default66.262.464.33.7

⚠ the two primers melt 5.3 °C apart — one annealing temperature will suit one of them better than the other

qPCR master mix: Owczarzy 2004/2008 · 250 nM primer · 50 mM Na⁺ · 3 mM Mg²⁺ · 0.8 mM dNTP

Primer-BLAST default: SantaLucia 1998 salt corr. · 50 nM primer · 50 mM Na⁺ · 1.5 mM Mg²⁺ · 0.6 mM dNTP

Bold, underlined base = 3′ end (polymerase extension point).

Primer QC

GC% · Forward

68.4%

GC% · Reverse

47.8%

Self-dimer ΔG · Fwd (any position)

-0.3 kcal/mol

Self-dimer ΔG · Fwd (3′ end)

0.5 kcal/mol

Self-dimer ΔG · Rev (any position)

-0.6 kcal/mol

Self-dimer ΔG · Rev (3′ end)

0.5 kcal/mol

Heterodimer ΔG (F×R, any position)

-2.1 kcal/mol

Heterodimer ΔG (F×R, 3′ end)

-1.6 kcal/mol

ΔG at 37 °C · SantaLucia 1998 NN parameters · < −6 kcal/mol = significant dimer risk · "3′ end" is restricted to duplexes anchored at a primer's own 3′ terminal base (polymerase-extendable); "any position" is the most stable duplex anywhere in the primer. Estimated screening value. For comprehensive dimer analysis, validate with Primer3.

Source

PMID

36504811(Europe PMC)

Citation

Xu et al. 2022

Genomic mapping

Ensembl gene

ENSGALG00010023764 ↗

NCBI Gene ID

769735

Assembly

bGalGal1.mat.broiler.GRCg7b

Cross-check

★ = Ensembl canonical transcript (reference model, not necessarily the most expressed isoform)

Showing: ENSGALT00010057941 ★

157bp1789bp117bp2782bp122bp3665bp123bp4438bp141bp5FRPredicted from the amplicon's own GC%+length (von Ahsen et al. 2001, at the same qPCR buffer as the primer Tm above: 50 mM Na⁺ plus 3 mM Mg²⁺ less 0.8 mM dNTP, as a sodium equivalent), not the SantaLucia model used for primer Tm, which isn't valid at this length. A screening estimate for ranking amplicons — that relation was fitted on oligonucleotide duplexes, so applying it at amplicon length carries more error than its published ±1.9 °C, and it is not a melt-peak prediction.204 bp amplicon · predicted amplicon Tm 88.3 °C
COX5A · ENSGALT00010057941 · canonical⚠ Exon-junction spanning · gDNA product still possible (300bp–1.0kb intron)

Off-target specificity

Specific

No paired forward+reverse off-target amplicon found against nt (whole genome/transcriptome). Screen requires >=2 total mismatches including >=2 within the last 5bp at the 3' end to clear a hit (NCBI Primer-BLAST-style stringency); hits with >=6 total mismatches are dropped as implausible.

Efficiency measurements

Ideal range:Dilution series 1.90–2.10·LinRegPCR 1.70–1.90·unknown method: not scored
No measurements yet for this primer pair.

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