Glucose in alkaline lysis! (was: Re: Merlin maxiprep help?)
(by ucgatan from ucl.ac.uk)
Tue Jul 24 06:15:55 EST 2007
On Mon, 23 Jul 2007, DK wrote:
> In article <Pine.GSO.4.58.0707231837160.26412 from socrates-a.ucl.ac.uk>, Tom Anderson <ucgatan from ucl.ac.uk> wrote:
> >On Mon, 23 Jul 2007, DK wrote:
> >> In article <vsbni.146$BJ1.116 from newsfe03.lga>, dk from no.email.thankstospam.net
> > (DK) wrote:
> >> >Looking at the maxi prep protocol reminded me of the reason there is
> >> >glucose in the cell resuspension solution.
> >> >
> >> >This can be a fun little puzzle:
> >> >
> >> >Do YOU know why 50 mM glucose is included in "P1"?
> >> OK, here is the answer: glucose has pKa at 12.5 (I think it is its
> >> aldehyde group), so glucose inclusion provides protection against
> >> irreversible DNA denaturation during alkaline lysis step. This is
> >> particularly important in large scale preps because, due to
> >> viscosity, mixing is very difficult and good neutralization without
> >> shearing chromosomal DNA can take very long time.
> >Interesting idea. Not to disrespect your authority, but do you have a
> >citation for that?
> Nope, I don't - since I never doubted the correctness of the answer, I
> never tried to track down the original reference. To me, it just makes
> absolutely perfect sense in every respect.
> That glucose (unlike, say, fructose or sucrose) has pKa somewhere in
> very high range, that I know for a fact.
Rohrer and Olechno, in Anal. Chem. 64:914 (1992), quoted in , give the
pKas of the monosaccharides as:
So you're quite right that glucose has a pKa up there, but actually,
fructose or galactose would have done too. Glucose is, of course, much
more likely to be to hand than the others in most labs.
The lysis buffer is 200 mM NaOH, which should, if i can do sums right,
have a pH of 14.7 - although i haven't accounted for the effect of the
SDS. That certainly fits your story from a pH point of view too.
At what pH does DNA undergo irreversible denaturation?
The NaOH is at 200 mM; the glucose is at 50 mM. Its buffering activity is
presumably called on when there is uneven mixing - is a few mM of glucose
going to be enough to hold 200 mM of NaOH below the critical pH?
> > It has a faint whiff of urban legend to it ...
> Why? What exactly bothers you?
This is not a very scientific answer, but gut feeling :/. I'm not saying i
think it's wrong, just that i remain to be convinced. An acceptable
attitude for a scientist, i hope!
Tom Anderson, MRC Laboratory for Molecular Cell Biology, UCL, London WC1E 6BT
(t) +44 (20) 76797264 (f) +44 (20) 76797805 (e) thomas.anderson from ucl.ac.uk
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