Black DNA from earthworms
afc at gnv.ifas.ufl.edu
Wed Jun 22 08:28:50 EST 1994
In article <54859.sjames at miu.edu>, <sjames at miu.edu> writes:
> I would like to know if anyone has encountered a similar phenomenon with
> other critters. When we do any of several standard DNA preps, we get a
> very dark brown to black pellet that turns out to be DNA- it looks like
> DNA to UV, it does ok when we try to amplify various genes, etc. Why the
> concern if it works? It doesn't always work. Some worms yield darker DNA
> than others, and there have been problems getting PCR reactions to go.
> Magnesium manipulation sometimes helps, sometimes not.
> It would be nice to know 1) what is this dark stuff, 2) how to get rid
> of it, or 3) how to consistently work around it. Yes, we have taken the
> dirt out of the worms first. Thanks to all, and I hope this is not a FAQ.
> Sam James 515-472-1146 fax:515-472-1167 sjames at miu.edu
I have worked with lots of different insects, and usually get colored DNA.
Some of them are quite pretty: Drosophila adults give red pellets, Lucilia
adults give yellow pellets. Mosquitoes give very dark purple to black
In all cases this just seems to be the result of some of the usual insect
pigments copurifying through ethanol precipitation and organic extraction
(CsCl gradients do get rid of them). The reds and yellows are pteridines,
some of the eye pigments. The purple seems to be anthocyanins. I would
expect that these or related compounds (e.g. a multiring structure with some
polar groups on it) are what you have.
I have not seen any effect of any of these on enzymatic reactions, so we
ignore them. My guess is that your intermittant problems are associated
with some other contaminant, perhaps detergent or organics from the isolation
or some negatively charged polysaccharides.
A few suggestions: 1) CsCl purify all of your DNA. Probably not worth it,
but it *will* work. 2) Run your DNA over a column. Depending on the resin,
it may get rid of some of the problem. 3) Add spermidine to your reactions.
More information about the Methods