I've seen some people post about this problem, but I too am having issues with low qPCR efficiency. I'm trying to develop a method for quantification of the A subunit of a functional gene called hydrazine synthase (hzsA). The primers I am working with were published in January 2012 and are hzsA_1597F ( 5′-WTYGGKTATCARTATGTAG-3′ ) and hzsA_1857R ( 5′-AAABGGYGAATCATARTGGC-3′ ).
I created my standard using the TOPO TA cloning kit for sequencing, digested the plasmid with EcoRI, and did a purification using QIAGEN Nucleotide removal kit. I quantified the DNA using a NanoDrop fluorospectrometer with the Quant-iT PicoGreen dsDNA Assay Kit.
I ran 10X serial dilutions of the DNA and used QIAGEN Quantitect SYBRGreen qPCR mastermix with 0.5 uL of 25 mM MgCl2 stock solution and 0.5 uL each of 15 uM forward and reverse primers in a 25 uL reaction. This reaction gave me a 68% efficiency. The melting temperature was 55 degrees C.
I did a preliminary experiment with 3 of my standards (10^6, 10^5 and 10^4 copies/uL) and increased the Mg concentration to 1 uL of 25 mM stock and got 95% efficiency, but when I ran the entire curve with the same conditions, I got about 78% efficiency.
Does anybody have any idea why my efficiency is so variable? And what can I do to stabilize it?