Hi everyone,
i recently did a qPCR to check the best annealing temperature for my primers. So i ran two of my primers on annealing temperature 59C with a melting curve. The melting curve shows only a single clear peak which suggest there is no primer-dimer formation. However, when i ran the product on agarose gel i saw very very faint band below my ladder that might be primer dimer. I also used different exposure time 300ms and 600ms and in the 300ms the band is almost non visible but in 600ms is slight more visible so i am slightly confused. The ladder i am using is 25bp ladder and my product size is 139bp and 90bp respectively. I have attached the picture of the gel and also the melting curve. Thank you
Hi, I think it's just the residues from your master mix like excess reagents. However, you may use a PCR purification reagent in order to see the bands clearly :)
Hi Scott
I don't think you need to worry about this!
Your melt curve looks perfect.
Your gel picture shows a very faint smudged band much below the 25 bp ladder mark. The chance for it to be a primer dimer is very less. This could be merely due to excess use of master mix. There is nothing to worry about this if your primers for Real-time PCR.
Cheers
This melt curve looks great to me. I think the smear you can see on the gel is probably unincorporated primers. If you wanted to confirm that, prepare a non-template control reaction and don't do PCR (no thermal cycling) - just run it directly on the gel. If the intensity of the smear looks the same or brighter in a sample which has never been heated, it's either unincorporated primers, dNTPs, or other signal coming from your master mix :)
i think that you are just seeing single primers not dimerised. possibly using too much primer in the pcr. It might be worth using less primer if it was a problem but the melt curve is good so it is not really a problem
There is no problem, as the melt curve looks good. This may be due to excess reagents in your reaction like excess primers, unincorporated dNTPs, and etc.
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