Glygogen/Microcons and disappearing DNA

Scrimshaw, Brian J SCRIMSHAWB%GRIEHFS at LINCOLN.CRI.NZ
Mon Apr 18 19:50:06 EST 1994


I am trying to get DNA from single hairs. I use the same protocol
as Higuchi in 1988 Nature. Basically a vigorous proteinase k
digestion followed by a single phenol/chloroform step, butanol
and concentration in a microcon 30.

I started this about 12mths ago and found I got better yields
when I put glycogen in prior to spinning in a microcon. I haven't
done this for a while, and went back to extracting from hairs in
the last few weeks.

To my surprise I got no amplifiable DNA, even from hair with a
root (positive control) The amplification is working ok since I'm
getting other DNA to amplify ok. I have new phenol, so it may be
that that's thye problem, and I'll test it today, but I got a
surprising result yesterday. I put 10uL of a mouthwash extract
(always works) with and without glycogen in a minicon, added
water and spun. I amplified the retentate and to my surprise
discovered that the DNA without the glycogen amplified, whilst
the DNA with the glycogen didn't. Does anyone in netland have
experience with glycogen and mini/microcons? Does glycogen
somehow stick to both the DNA and the filter? I don't understand
it. I'm sure I haven't mixed up my samples, but will repeat the
experiment today with a bunch of DNA samples to see what happens.

Any help or protocol advice appreciated.

Brian Scrimshaw
Wellington
New Zealand



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