I am evaluating a primer set that was designed by previous research. I am using Quantitect SYBR Green (Qiagen), the template volume is 5 uL ( total reaction volume 25 uL). I run triplicates starting from 10^8 to 10^1 copy plasmids/mL. The linearity range is 10^8 to 10^4, and below 10^4 the Ct is stagnant already. I have tried using 10ul for the volume of template, the Ct shifts higher but the limit of detection stays the same as the previous run.
I am confused with the poor LOD of this assay. Can I try something to increase the detection limit at least until 100 copy/mL?
does this also mean that this primer design isn't good? or the stagnancy of Ct below 10^4 copy plasmid/mL is due to stochastic effect of PCR?
From my understanding, your lower LOD is 50 copies (10^4*(5 uL/1000 uL)). Fifty copies is already quite low, but if you are trying to reduce even further, I would recommend adding 2-5% DMSO to the reaction. This should help increase primer annealing efficiency at low template concentrations.
Your >100% efficiency at high template concentrations indicates there might be a polymerase inhibition problem. Have you checked the purity of your samples? You're using a large volume of template, so it makes sense that there might be an excess of inhibitors.
Vanessa Smilansky Thanks for your response, the purity of the plasmid is 1.99 (A260/A280) and I'm actually a bit concerned because I know that the best is 1.8. But I also find several literature stating that as long as the purity is 1.8-2.0 it is okay to be used as DNA template. Do you have any opinion about this?
I agree that purity should be OK. Still, you could try concentrating your template so you don't have to start with such a large volume for your dilution. Or try using a purification kit. Recommended amplification efficiency is between 90-110%, so it is worth trying to reduce if you can.
Vanessa Smilansky I want to know too, if I make 2-fold dilution series (1:2) should I use the same efficiency formula (E = -1+10(-1/slope)) or is the formula used depends on the dilution factor we use? I've read several QnA session that if we use 2-fold dilution series we must change the equation into E = 1+2(-1/slope)
I believe for a 1:2 dilution you would use the formula E = 2^(-1/slope)-1, and you would be aiming for a slope around -1 (i.e. each dilution should be 1 cycle apart, as opposed to 3.3 cycles in the case of 1:10 dilution)
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Vanessa Smilansky I will try again for the standard curve tomorrow, I will make two fold dilution series starting from 10^6 to 5.10^2 copy/mL and use 7,5 uL template volume out of 25 uL total reaction volume (instead of 10 uL template volume). Wish me luck :")
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