It's an end-point PCR protocol. I'm using 1.5% agarose gel with SyBR Safe dye and TBE as a running buffer, visualization on BioRad XR+ system.
I was primarily thinking of primer efficiency, whether the pairs producing the shorter amplicons are less efficient in a multiplex setting? Because when placed separately, the amplicons produce deep bands.
Please do suggest, I'll appreciate your insights!
Typically, the shortest amplicons will have preferential amplification - with the HUGE assumption that all primers are equally efficient. Like when you use primers for Insertion/Deletion detection. The shorter product will amplify more than the longer.
It is very challenging to get multiplex genotyping to work. You can test out various concentrations of each set of primers, different annealing temps, etc.
Honestly, unless you really need to develop a multiplex genotyping reaction, it might be faster & more reliable to set up single reactions.
Good luck!
Are you getting a low molecular weight fuzzy band badly defined and around 80bp in size . If so you may have a primer dimer problem removing primer from the shorter product pcr reactions and, if so, using a hot start enzyme might help and at least you have the single plex reactions to keep the data flowing while you sort this out
Those band sizes are not so distinct to clearly be visible of normal agarose gel. Further, band band migration is not the same as you think it would be. The lower band might still be longer fragment, becuase most probably you are directly loading the PCR product on gel without quantifying the ng DNA per µl.
On your single band has all the three DNA lengths, most probably this is what happening.
In conclusion, it can be said that the band difference can not be good if the sizes are so close to each other.
Abhijeet Singh Thanks for pointing out this interesting insight. We ran a real-time PCR with the same design to verify this issue. However, we are seeing a sharp, single peak (interpreted as a single amplicon) in the melt-curve analysis, as well. We're running this HRM analysis with an increment of 0.1 degrees Celsius.
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