Hello John,

All commercially available kits should be sterile so it is hard to believe it is the kits themselves. Most often when you are getting background contamination in extractions it is from the reusables (i.e pipettes, sharps, bench-tops, etc.) or from the sample itself. For example when we were extracting DNA from a snail specimen in the past we had issues with a contaminate. When we sequenced the contaminate it turn out to be an algae species that was forming a thin film on the shells that we did not notice before. After that we started to remove the snail from the shell and the contaminate issue went away.

If I would you I would start by making sure that all of the reusables are properly sterilized. If the contamination still persist then you can sequence the contaminate (i.e. COI is what we used in the snail example) and see if you can figure out what the contaminate is.

Hope this helps.

Best Regards

Artur and Max: Like you, I had assumed that biochemical reagents and DNA extraction kits did not contain background DNA, but unfortunately they do. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4882852/

See the article in the link above for a recent discussion of this issue. I my studies I have used the ZYMO DNA extraction kit and performed experiments that included sterile TE buffer or PBS solution processed through the DNA extraction kit/process, as well as analyzing the TE buffer and PBS solution without any processing (no exposure to the DNA extraction kit). All the samples were then included in qPCR assays, along with no template control reactions, to detect total bacteria using universal primers targeting the 16S rRNA gene. I have tested this several times and I consistently find that TE and PBS samples processed through the DNA extraction kit yield lower Ct values than the unprocessed TE and PBS samples. The reagents in the DNA extraction kit are contributing trace amounts of background microbial DNA, and the literature supports my conclusion.

My question is, have other researchers found that some commercial DNA extraction kits have lower levels of contaminating bacterial DNA?

If the research community is not aware of this DNA contamination issue then it is important to spread the word and find solutions.

Regards,

John Kilbane

As the author of the article cited, I can tell you that all reagents have ample amounts of bacterial DNA. Even treating with UV, there is enough bacterial DNA in other reagents that cannot be irrdiated (like polymerases) to make up for it. Irradiating all components does seem ro reduce the amount of DNA, but not diversity. It is important to monitor your contamination so that you do not find something unusual only to later determine it is a contaminant. There are NO CLEAN REAGENTS.

Another useful article I found on this topic is titled "Reagent and laboratory contamination can critically impact sequence-based microbiome analyses" and it  can be accessed using this link 

http://download.springer.com/static/pdf/253/art%253A10.1186%252Fs12915-014-0087-z.pdf?originUrl=http%3A%2F%2Fbmcbiol.biomedcentral.com%2Farticle%2F10.1186%2Fs12915-014-0087-z&token2=exp=1474911861~acl=%2Fstatic%2Fpdf%2F253%2Fart%25253A10.1186%25252Fs12915-014-0087-z.pdf*~hmac=b22fdaa9cd19e799c67af2a83ea85ca298ee6af7fbf82e6fdba89fde574c69d0

This study found microbial DNA contamination in all the commercial DNA extraction kits tested, and samples having low DNA concentrations were most vulnerable to problems with contaminating DNA. This study also indicates that the commercial DNA extraction kit with the lowest background (among the several tested) was the MoBio Ultra Clean Microbial DNA Isolation Kit.

qPCR or DNA sequencing of environmental samples with low DNA concentrations need to beware of the effects of contaminating DNA on any results obtained.

Regards,

John Kilbane