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16s double bands obtained-why?? - (May/31/2012 )

Hi everyone,
I have been having a problem with 16s sequencing when trying to identify a strain.
I have 16s primers designed for Lactobacillus that I use quite routinely (have screend ~30Lacs with these) without any issues, I always get a good clean band at the expected size. However, I have a strain that is giving me a 16s band and then another (failry weak) band just underneath it. I though this might mean I have contamination in my stock but I have plated this stock out numerous times now and I have never seen anything else there. I have sent the sample for sequencing anyway and it comes back a Lactobacillus but the quality of the sequence is very poor and the company I use have suggested it looks like a mixed template. I have picked numerous individual colonies and they have always come back as a Lactobacillus. Could there be a second Lac entity in there too?
Any suggestions are greatly appreciated, thanks!


could be mispriming (u use gDNA as a template) ...i would not worry to much.



Ok thanks, will try gDNA so!