16S primers can amplify DNA from pretty much any sample (bacteria, vertebrate, plant, etc.), even if they are supposed to be optimized for say fish vs. bacteria.

Sequence a few and see what comes back!

Hi Alex,

please check out the publication of Palumbi et al. 2002

PALUMBI S.R.,MARTIN A.,ROMANO S. ,MCMILLANW.O, STICEL. and GRABOWSKIG. 1991.The simple fool’s guide to PCR. Version 2. University of Hawaii Press, Honolulu: 45 pp

(you can find the PDF here Simple_Fool's_Master PCR.pdf)

I don't know whats about the year of the publication - there was a version one from 1989 (however I have seen also citations for version 2 with the

year 1991 and found also a version one from 2004 ).

Nevertheless, in this publication describes the primers you are using.

This is what they say about the primers "These 16s primers work for urchins, vertebrates, insects, gastropods and just about anything else. The 16sar and 16sbr (the complements of 16sa and 16sb) primers face one another and can be used to amplify a 500-650 base fragment of the 16s RNA coding region from just about anything."

For me this does't sound like these primers are vertebrate specific (see Katie A S Burnette answer) and I would conclude that you get multiple bands from different organisms - as Katie suggested as well. And I think it it always a good idea to search for the original publication (even if this is somehow weird in this case):-)

I hope this gives a little more insight

Hi Alex

Try gradient PCR to get the optimal annealing temperature where you will have a single band

Hi! That’s great that you’re going to sequence that unknown band. I’d recommend sequencing both bands to make sure your target is actually the one you think it is. On another note, to favor smaller fragments, you can shorten the polymerization time—this way, the smaller fragment is more likely to be amplified. I hope your experiment goes really well. Best regards!