Carbohydrate against DNA summary
hiesi at mol.univie.ac.at
Fri Nov 25 07:46:10 EST 1994
Thank you for your help and all your ideas.
Some answers have been sent to me by E-mail. I thought that the replies
will be also interesting for other people.
>I like to purify DNA out of chicken egg yolk. I always end up with a
>stuff that looks and behaves similar to DNA but is not DNA. I think it is
>a carbohydrate. I wonder if somebody could give me an idea how to remove
>Now more exactly: I dilute the yolk and make an over night Protein
>degradation with proteinase K. Then I perform a Phenol/Chloroform
>extraction. After the precipitation with isopropanol I get this white
>huge pellet which is not DNA. This Substance cannot be removed with
>Phenol, not digested with amylase or with hyaluronidase. It is water
>soluble (better soluble in acidic solution), makes a kind of gel in basic
>solution. And makes me nervous. What is it? How to remove it. I would be
>grateful for any help
>Institute for molecular genetics
Even if you remove the mystery glop, one non-fertilized egg (=one cell)
should have only about a pg of DNA in it. What are you planning to do
with it? If you just need chicken DNA, why not consider blood? Red
cells are app. 5 exp9 cells/ml, and are nucleated in birds, so one drop
(say 50 ul) should have several ug of DNA.
If you need the ovum DNA, I guess you should probably throw away the yolk
and try to find the nucleus, if possible, I'm not a developmental
biologist, so I can't tell you how to do this, though.
Unfortunately I do not work with chickens but instead I work with trees
.......anyway when I extract DNA from conifers many times starch and
are associated with the DNA. The best way to remove them I find is to
a spermine ppte.
The proceedure that I use is based on a method published in Nucleic Acids
volume #9(20)(1981) by Barbara Hoopes et al. pages 5493-5504 called
of DNA preciptitation by Spermine". After the isopropanol ppte.
DNA in a suitable
volume of TE and then follow the directions in the paper. I have also
another method to remove
unwanted junk from my DNA; a method called differential salt ppte.
you ppte. your
DNA in high salt conc. followed by a second ppte in lower salt conc.
this method never
seemed to work for me. Getting back to the spermine ppte. other people
work in flies say
that a spremine ppte.is the best way to remove any kind of junk from DNA
regardless of organism.
Anyway feel free to e-mail me if you have any questions about this stuff.
You could use Cetylmethylammonium bromide from Sigma #5882,
which you can see in one article of M.G.Murray and W.F.Thompson
volume 8 number 19 1980 Nucleic Acids Research how to do.
You also should check Methods in Enzymolozy vol. 152
Dear Thomas -
2 thoughts on your problem:
My first reaction is that the white substance could be NaCl, which
coprecipitates w/DNA in isopropanol. Try using ethanol and see if it
disappears. (Just an idea - I can't explain why it is more soluble in
Secondly, have you considered a CTAB (hexadecyltrimethylammonium bromide)
extraction? This is good at getting rid of polysaccharides. After
proteinase K digestion,
1. bring the [NaCl] to 0.7 M
2. add 0.1 volumes of (10% CTAB in 0.7 M NaCl). Heat at 65 degrees C for
3. CHCl3/IAA extract
4. phenol/CHCl3/IAA extract
5. ppt the DNA - note the high [NaCl], so you'll need to do a couple 70%
The lipids in the egg do tend to give funny reverse phases. You
might want to try and extract the lipids out of your system with
twice before using isopropanol. I hope it works. Good Luck!!!!!
There is so little DNA in an egg yolk that I doubt you will ever be
successful. An egg contains a single diploid or tetraploid nucleus,
so even with 100% recovery you would end up with a few picograms of DNA
per egg. In any case, what you describe sounds like polysaccaride, many
of which co-purify with DNA, and are not removed by any of the
techniques you describe. I don't know how to get rid of polysaccaride;
some polysaccaride contamination is not usually a problem, but then most
applications don't have your problem that you are trying to recover
picograms of DNA from an enormous mass of tissue.
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