Problems with dissolving genomic DNA

Bruce Roe broe at aardvark.ucs.uoknor.edu
Wed Jul 14 07:25:00 EST 1993


In article <21v58g$sma at samba.oit.unc.edu>, plc at med.unc.edu (Philip L. Carl) writes...
>I have been having a problem dissolving genomic DNA prepared
>by the method given by Sambrook et al., Molecular Cloning
>Second Edition, Cold Spring Harbor Press, 1989 pp 9.22.  The
>method is based on the guanidinium HCl method of Bowtell,
>Anal. Biochem. 162: 463 (1987).  The method seems to have a
>lot to recommend in that it is fast, cheap, uses minimum
>hands on time and is reputed to yield DNA of greater than 80
>Kbp.  Bowtell says the DNA may be somewhat harder to
>dissolve in TE than DNA prepared by the phenol, SDS,
>Proteinase K method and Sambrook at al. warn against letting
>the DNA dry too much prior to dissolving.  Even taking these
>considerations into account, I can only dissolve the DNA to
>about 45 ug/ml and that only after heating to 370C
>overnight.  This is really too low to be convenient for
>Southern blotting which is what the method is designed for.
>Bowtell says the DNA is dissolved at about 0.25 ug/ml
>(probably a misprint for 0.25 mg/ml) and Sambrook et al.
>imply the DNA is soluble to at least 70 ug/ml.  The only
>significant difference between the original method and the
>adaptation in Sambrook et al. is that Bowtell washes the DNA
>in 70% ethanol while Sambrook et al. use 95% ethanol.  I've
>tried it both ways, and still have problems getting the DNA
>to dissolve.  Has anyone out there tried this method who can
>offer hints as to how to avoid isolating insoluble glop?
>Please reply by the Net or E-mail to plc.unc.med.edu.
>Thanks.

Try placing the tube in the cold room on a rocker or shaker that
can be adjusted to VERY GENTLY rock the tube overnight.  Vigorous
mixing will shear the DNA more than you may want.

As for the drying, if you dry a pellet in, for example, a SpeedVac,
you'll get a pellet that will not have a very large surface area
and thus be difficult to dissolve.  Thus, by evaporating off the
ethanol and most, but not all of the water, usually will allow the
DNA to dissolve more quickly.

As for the 70% vs 95% for the wash step, you'll just have to do the
expt.  70% has been used because of the increased solubility of phenol
in 70% rather than 95% (at least that's what is thought, havn't done
the direct expt ourselves).

We use a 70% wash and don't dry too much and then store in a ice bath
with perodic gentle mixing until all is dissolved.

Cheers......bruce
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