I’m experiencing issues with low and inconsistent qPCR efficiency while developing a quantification method for the A subunit of the hydrazine synthase gene (hzsA). I’m using primer pairs from a publication:
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hzsA_1597F: 5′-WTYGGKTATCARTATGTAG-3′
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hzsA_1857R: 5′-AAABGGYGAATCATARTGGC-3′
To create my standard, I used the TOPO TA cloning kit for sequencing, digested the plasmid with EcoRI, and purified it using the QIAGEN Nucleotide Removal Kit. DNA concentration was determined with a NanoDrop fluorospectrometer using the Quant-iT PicoGreen dsDNA Assay Kit.
For qPCR, I prepared 10-fold serial dilutions of the DNA and used QIAGEN QuantiTect SYBR Green qPCR Master Mix with the following additions in a 25 µL reaction:
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0.5 µL of 25 mM MgCl₂ stock
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0.5 µL each of 15 µM forward and reverse primers
Under these conditions, I obtained an efficiency of 68% and a melting temperature of 55 °C.
In a preliminary test using only three standards (10⁶, 10⁵, and 10⁴ copies/µL), I increased the MgCl₂ to 1 µL of 25 mM stock, which improved efficiency to 95%. However, when I ran the full standard curve under the same conditions, the efficiency dropped to ~78%.
Does anyone have insight into why my qPCR efficiency varies so much, and what steps I can take to make it more consistent?