Sequences
Forward (5′→3′)
TGAGAGCATTTGGACGGAGGReverse (5′→3′)
TTAAATCCTGGATGGTCGGCAAmplicon
135 bp
estimated from mapping
Primer Tm (°C)
| Conditions | Forward | Reverse | Average | Δ F/R |
|---|---|---|---|---|
| qPCR master mix | 63.5 | 63.0 | 63.2 | 0.6 |
| Primer-BLAST default | 59.7 | 59.4 | 59.6 | 0.3 |
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
55.0%GC% · Reverse
47.6%Self-dimer ΔG · Fwd (any position)
-0.3 kcal/molSelf-dimer ΔG · Fwd (3′ end)
No duplex foundSelf-dimer ΔG · Rev (any position)
-2.0 kcal/molSelf-dimer ΔG · Rev (3′ end)
0.5 kcal/molHeterodimer ΔG (F×R, any position)
-1.1 kcal/molHeterodimer ΔG (F×R, 3′ end)
-1.1 kcal/molΔG 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
Citation
Siddique et al. 2022Genomic mapping
★ = Ensembl canonical transcript (reference model, not necessarily the most expressed isoform)
Showing: ENSMUST00000006949 ★
Off-target specificity
SpecificNo 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 scoredAnalyse your qPCR run with LinRegPCR efficiency correction and a MIQE 2.0 report.