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Old 04-29-2015, 02:38 AM   #1
sparky
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Location: Australia

Join Date: Mar 2014
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Default DNA degradation - gill tissue

Hi all,

I was wondering if anybody has experience extracting DNA from fish gills (and the microbes associated with them)? I have been trying to extract genomic DNA from the gills of some fish heads that we obtained from a local fish market. When I run a gel to check the yield/quality of the DNA, there is no hmw DNA visible and little or no lmw DNA (<100bp). I have seen in the literature that DNA has been successfully extracted from canned tuna and from other retail fish sources.

I was expecting to see some evidence of DNA degradation, as we do not know how long the fish had been dead for before we sampled the tissue, but it seems strange to me that I did not see any larger DNA fragments whatsoever. I would have thought that I would have at least got something out of whatever live microbes were associated with the gills at the time of sampling? Sampled tissues were stored at -20C. I have repeated the extraction with tissue homogenisation performed on ice and have included a control sample of Drosophila adults to confirm that our reagents etc are ok.

I know that samples like this can be tricky to work with, and often have a high load of DNases, but it seems unrealistic to me that all the DNA would have degraded, especially given that DNA is widely used in forensic biology?
I would be grateful to get people's opinions this.

Thank you
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Old 05-07-2015, 11:12 AM   #2
pmiguel
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Location: Purdue University, West Lafayette, Indiana

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Default

Do you pre-stain (include the dye in the agarose) or post-stain your gel? If the former it is possible that you just have a lot of degraded DNA, or RNA, that effectively "de-stained" your gel as it ran, sweeping away all the dye, leaving none to bind higher molecular weight DNA. However this is only likely if the low molecular weight stuff looks very bright.

But, also, if your DNA prep doesn't do a good job of inactivating all the DNAses in the gill cells, all your DNA may, in fact, have been degraded.

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Phillip
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