I'm working on the 16S rDNA amplification of cow milk microbiota. For the last few months, I have been trying different primers and optimizing temperatures. It seems like I always have some smears or "parasite" bands on my gel migration afterwards. Is it something common in such qPCR amplifications?
What is size your ladder?
It seems you have primer dimer. It is better you try a gradient PCR at firs to optimize your temperature, and so concentration of your agent such as primer.
It's a 2 Log DNA Ladder (I linked it below here). I also tried at different annealing temperatures (linked below as well). In 1 it's genomic DNA from S. aureus Newbould and in 2 it's DNA from a milk sample.
It is look like your temperature about 60 is good.
But you check the concentration of extract DNA?
There is many factor can be important, concentration of your DNA, concentration of primer that you added and concentration of MgCl2.
If you optimize those you have not smear or extra band.
The 16s rRNA gene is rather a complex target as it is full of internal homologies, so I'm not surprised you're getting some extra bands. There are also compromises in the design of the primers in order to try to achieved universal taxonomic coverage.
You can spend months optimising PCR reactions, trying to find the sweet spot. Unfortunately, if the sweet spot is hard to find, it's likely to be quite narrow, so it's easy to drift off it again. That said, you might like to try primer titration - running various combination of primer concentrations; the standard is 900 nM, 300 nM and 50 nM, though I think the last is a bit low. This helps to compensate for differences in stability between the two primers.
But if you just need a clean band, you may save yourself time by cutting it out of the gel and eluting it. The drawback here is that it involves a lot of post-PCR manipulation and produces a lot of contaminated surfaces - including your skin, hair and clothes.
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