I am performing a standard curve before relative quantification using Roche SYBR GREEN 1 master in LC480 (Roche) and 6 dilutions of cDNA in triplicate. For some of my primers, I get one peak in the melting curve (I am not totally sure, I have attached 3 examples here) and also the gel shows one single band for my target amplicons. However, when I calculate the Efficiency, It is 94% and 134%.
I should mention that sometimes I get primer dimer peak for my NTC.
I would be appreciated if you can explain what could be the possible problem here?
(is it my primers, annealing temperatures, my template or master mix etc?)
Thank you in advance.
Hi Nahid,
there are multiple ways to optimize your PCR reaction. In which Ct-range are your signals? Depending on how many targets you want to measure it can be quiet time consuming. The first thing you could do is to up your TM when performing a 3-Step qPCR. This might help to avoid unspecific primer binding and dimerization. Then you can play with your primer concentrations as well as with the amount of your input cDNA. Or you can design whole new primer sets alltogether.
Best regards
Hi Soner Öner-Sieben ,
Thank you for your answer.
Ct values for my HKG is 15-27 and for my GOI is around 26-36.
The TM of my primers is around 60degC and I used 60degC as TA. However, a TA of 55degC worked for the other two primers. Now I am not sure if 55degC would work for the rest of my primers.
I am doing 3-step qpcr. Do you mean to increase the annealing temperature? Should I set the TA at higher than 60degC?
Hi Nahid, you should perform a gradient PCR to determinte optimum annealing temperature, you can do this with conventional PCR since the gel electrophoresis will allow you to see the formation of primer dimers on your sample on the given temperatures. I used to have primer dimers formation and I solved it by rising annealing temp and decreasing primer concentration.
Hi Franco Matias Viscarra,
Thank you for replying.
Can primer dimer be a cause of low (or higher than normal) efficiency?
I will try to play with Ta and concentrations.
Yes, not optimal reaction conditions and concentrations are a common cause for bad primer efficiency and primer dimer formation. Another factor can be cDNA concentrations. BTW 94% it's a pretty good efficiency so you will be trying to fix the 134% one.
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