Carbos. in DNA prep!!!!!!
tchipman at is.dal.ca
Thu Aug 29 14:49:25 EST 1996
I've expericed similar problems with a number of preps (using field
collected fern, also with field-collected algae)
The most effective technique I have found for dealing with carbos in total
DNA preps from ferns was by adding 7.5M ammonium acetate to the aq.
material retrieved following a few phenol / organic extractions. Spinning
this for 15mins at 16,000g - this pelleted out LARGE gobs of
polysaccharides, and subsequently, DNA was precipitated with isopropyl
alcohol... etc.. etc. While previous DNA preps NOT incorporating this
step generally gave a reasonable yield of DNA, the material was
indigestible to a selection of restriction enzymes. However, when I
incorporated this step into a rather standard DNA isolation procedure, the
material was of good yield and good (digestable) quality.
More specifically on the protocol,
-Grind tissue in buffer/detergent, perform organic extractions (I usually
do a few passes of CHCL3/Isoamly at this point), then: add 1/2 volume of
7.5M ammonium acetate to the aq. from the organic extraction. Spin
15minutes at 16,000g. Drain off liquid, pellet (if present) should be
pretty solid. Alcohol precipitate DNA, continue on as you wish.
Alas, I don't have a terribly good reference for this protocol - I was
given the procedure by some colleagues here at Dal, and the one ref. they
cited didn't seem to discuss this application of NH4oAC at all.
One last note: I have also tried this on some freshwater algae, and
while I've had similar problems with polysaccharides, incorporation of
this step did NOT yield a carbo pellet, and it didn't make any detectable
difference to the quality of my DNA. However, the amount of carbo.
present in the two cases is significantly different - the fern preps,
upon grinding of tissue, were EXTREMELY "Slimy", whereas the algae preps
are not. Possibly this procedure is most suited for extreme cases only,
I don't know.
Hope this is of some use,
Rodney Earl Pettway (REPE at gnv.ifas.ufl.edu) wrote:
: Could someone please help me. I am using a DNA extraction method that
: uses CTAB/Sorbitol/sarkosyle as detergents. In the tube that has my
: DNA in it there is always this bellet at the bottom. It looks loke a
: carbo pellet. I has tried everything to eliminate this from my prep.
: Please help.
: Thank you
: Rodney Earl Pettway
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