Fw: appearance of "clean" DNA?

Deanne Bell dbell at qnis.net
Fri Jan 5 11:46:55 EST 2001


Hi Marc
I have found that most of the contamination left in my plant DNA preps is
due to polysaccharides.  Depending on the species you are working with
there may also be polyphenolics, lipids, . . . lots of other stuff.  I have
found that the basic CTAB isolation protocol does not always work with all
species.  I have one technique I use for isolating DNA from tumbleweeds,
another technique for grapes, another technique for prunus species, and
another technique for lemon flavedo and peppers.
Most references claim that a high salt wash will remove polysaccharides, I
am not convinced of this.
IMHO - I think that the polysaccharides are responsible the viscosity of a
prep in TE buffer - you MIGHT get a viscous sample if you have a grossly
huge amount of DNA in too little TE buffer.

I use about 75mg fresh leaf tissue and a final TE volume of 200ul.  In my
final ETOH ppt, I add sodium acetate and  ETOH.  With gentle inversion, I
see schlieren lines form and then the solution becomes viscous with
continued inversion.  The viscosity here makes sense to me because The DNA
is precip. out of solution.  I can actually tell the difference between two
tumbleweed species at this point because one of them will become cloudy,
milky white -  the other species will remain clear and colorless.  Both
preps are slightly viscous and give me good amplifiable DNA, so I haven't
tried to purify the preps anymore than this.

My supervisor and I have an on-going debate of what a good pellet looks
like.  Spin down the ETOH ppt and pour off the supernatant.  I have seen a
range of ppts: from a clear gelatinous smear down the side of the tube wall
to a hard white pellet at the bottom of the tube. If my fresh or dried
tissue is old or distressed, I even get dark brown/green pellets.  This
will usually clean up a bit with repeated ETOH ppts.

I have had a very hard time getting good, amplifiable DNA preps from Vitis
(grapes).  The final ETOH ppts are clear, colorless and slightly viscous
but there is some contamination preventing amplification.  So you can't
always tell that you have a really pure prep just by looking at the TE
buffer and ETOH ppt.

I run 3ul of my DNA prep out on a 0.8% - 1% agarose gel.  If I get a nice,
tight, high molecular weight band on the gel, I know I have good genomic
DNA.   My test of "purity" is repeatability in the amplification.  If it
amplifies well, then I don't need to mess with my DNA isolation protocol. 
If I don't get amplification, then I try other isolation procedures or try
getting my fresh leaf tissue early in it the growing season when the shoots
first break dormancy.

> Perhaps this is just a theoretical question, since it may not be
> possible to obtain completely clean genomic DNA, but I'm still curious
> what people think about it.

This is probably more than you wanted to know. But you said you were
curious :-)

Deanne Bell
Molecular Markers Lab Technician
USDA Agricultural Research Service
2021 S. Peach Ave.
Fresno, CA 93727
e-mail: dbell at qnis.net
phone: 559 453-3170
fax: 559 453-3088
----------
> From: mwcrepeau at my-deja.com
> To: methods at hgmp.mrc.ac.uk
> Subject: appearance of "clean" DNA?
> Date: Thursday, January 04, 2001 5:33 PM
> 
> What does clean genomic DNA look like?  It should, of course, form a
> clear, colorless solution when dissolved in TE buffer, but what about
> viscosity?  I remember a graduate student looking at one of my first
> plant DNA preparations when I was un undergrad and telling me that
> there was a good yield because the prep was very viscous.
> 
> Later, someone else told me that DNA itself is not viscous in solution
> and that any viscosity is due to contaminants.  That seems reasonable,
> but it has also been my experience that the viscosity is almost
> impossible to eliminate, even with several additional phenol/chloroform
> extractions and/or ethanol precipitations.
> 
> What is responsible for the viscosity that is typical of plant genomic
> DNA preps?  Moreover, if I could get my genomic DNA completely clean,
> so that nothing was left but the nucleic acid itself, what would it
> look like in solution?  And what would it look like as an ethanol
> precipitate?  Would it appear as the white cloud that I am accustomed
> to seeing, or is the white color due to bound protein and/or other
> contaminants?
> 
> Perhaps this is just a theoretical question, since it may not be
> possible to obtain completely clean genomic DNA, but I'm still curious
> what people think about it.
> 
> Marc Crepeau
> <mwcrepeau at hotmail.com>
> 
> 
> Sent via Deja.com
> http://www.deja.com/
> 
> 


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