Hi all,
I run a PCR to look for an integron in different bacterial isolates to test the presence of this integron in the bacterial genome. This integron is often associated with resistance genes. In your opinion, how should I interpret the attached image of the gel for this PCR ? I have clear bands for some isolates, but for others (e.g. well 2 8, and 9) I have a very faint band and other different bands. The faint band is of the same size of the clear bands, so I don' t know if it is really the gene or just an aspecific product. What about the other aspecific bands I have? Can they have something to do with the fact that the PCR product is an integron?
Thanks
Hi
Did you include a positive and negative control in the run? And the lanes are not labelled(PC NC, SAMPLES,etc). Label the gel lanes so that I can try..
Dear Silvia
I do not know, but it seems that the clear bands of lanes 2, 3, ... are so small (maybe 100-200 bp?) An integron containing at leasst one gene-cassette has at minimum >500-600 bp. If I am right, the most probable scenario is that belongs to empty integrons (It is a normal scenario when works with class 1 integrons). Presence of empty integrons may be relevant, because the bacteria has the machinery to incorpore antibiotic reistance gene-cassettes.
Regarding remaining bands, in my experience this type of thin repetitive band patterns usually are due to inespecificities, and to the lack of true integrons. I will send you a paper (Diagnostic Microbiology and Infectious Disease 48 (2004) 175–179) in which we found a similar scenario.
Kind Regards
Quim
PCR is not an "on at one temp off at another". It is constantly in motion and will bind non-specifically quite often. PCR operates on the hope that the specific product will be generated far more often than the non-specific.
That being said, just cut them out of the gel, clone them, and sanger sequence them if you're still curious as to what they are.
If you want rid of them, try a nested PCR, extending your primers and raising your annealing temp, or just raising the annealing temp.
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