I am facing problem in qPCR analysis. The file attached shows the melt curve of GAPDH. In my setting, my template was 100ng and primer concentration 500nM in 20ul. Cycling number was 40 cycles. In this run, I did for 2 other genes. Only my GAPDH have this problem. Ct value is about 25.9. I am suspecting primer dimer. However, 1uM of primer can give a nice melt curve. Why 500nM can give this result?
Your melting curve showed 3 sharp peaks. we can see small and broad peak near 65 ºC. It may related to primer-dimer (the lower temperature), we can also see another sharp peak near 75ºC and 88 ºc. You should if they are 3 pcr product in gel electroforesis. Should use biorad software or related to see well if the weak band appear in the gel. If you use silver green dye try to decrease concentration.
My primer is running out soon. Hence, I try to use lower concentration
Dear Lim,
I think, your total template amount and primer concentration is too high. Decrease your total template in per well. As it is fluorescent based quantification, huge amount of template sometimes gives you false positive result. I use only 2 ng of total template and 200 nM of primer for 10 μl of reaction. Kindly, once go for lower amount of template and i hope it will work.
Hi,
Try the above given modifications, just before doing it- u need to see the products on gel, which gives u a clear idea on what happened to ur reaction.
If im right I suspect a strong nonspecific product amplification along with the desired one and a dim primer-dimer in ur reaction by seeing that image.
Ct values caused by primer dimer alone can be neglected (usually they appear at 34 or 35th cycle in even with routine reaction component levels). For optimum quantitation of desired product, one must adjust reaction accordingly, each template type/primer system may have their own unique performances which is apparent.
While reducing the component levels (primer for example) in reaction, dont give a drastic change but go for maximum of 20% to 35% reduction in ur case by gradient setup at each level varied by 10%.
Do great, gud luck!
Hi! I recommend you to use less than 50 ng of template. Also, you can use between 200 and 1000 nM for each primer. It is puzzling to see that you got good results with 1000nM (This may happen when primers get old and degraded). If you don't need to maintain PCR temperature conditions use touch down PCR.
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