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estaunton 02-13-2013 08:18 PM

Low PCR efficiency
 
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?

namishu95 02-15-2013 06:06 PM

Re: Low PCR efficiency
 
First of all, the primers were with very low Tm ( forward primer even with 35⁰C of Tm )and several degenerate bases. second the PCR amplicon was a little longer, it is ideal for SYBR green assay to have 100-150 basepais amplicon.
the key of real time PCR is with great primers.

ajithnandanam 03-28-2013 06:53 AM

Re: Low PCR efficiency
 
PCR assay efficiency range for real time PCR assay is 90% - 110%. An assay efficiency above 110% indicates a possible inhibition in the real time PCR reaction. The main reasons for inhibition in the reaction is the poor quality of DNA or RNA used as template, use of high template concentration. Sub-optimized extraction procedures to get the DNA or RNA purified, presence of high amount of chaotropic salts which can inhibit Taq polymerase activity. Low efficiency real time PCR assay is mainly due to the poor reaction conditions or reagent concentrations which includes sub-optimized concentration of Primers, Probes, Taq polymerase, Magnesium, etc. and the reaction condition includes improper or sub-optimal thermal cycling.


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