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16s double bands obtained-why??


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#1 sillysal

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Posted 31 May 2012 - 05:25 AM

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!

#2 pDNA

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Posted 31 May 2012 - 09:26 AM

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

Regards,
p

#3 sillysal

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Posted 01 June 2012 - 04:17 AM

Ok thanks, will try gDNA so!




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