Problems with dissolving genomic DNA
chai_z at wehi.edu.au
chai_z at wehi.edu.au
Wed Jul 14 02:13:51 EST 1993
From: chai_z at wehi.edu.au
Subject: Re: Problems with dissolving genomic DNA
Message-ID: <1993Jul14.113820.1 at wehi.edu.au>
Date: 14 Jul 93 11:38:20 +1000
References: <21v58g$sma at samba.oit.unc.edu>
Organization: Walter & Eliza Hall Institute
In article <21v58g$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.
I experienced the same problem before. After lysis of the cells (tissue
powder), I added ethanol gently and picked DNA from the interface by rotating
a "U" shaped glass loop (made from a Pasteur pippete). Some white, strong fabre
materials were obtained very easily (good yield and good looking). I tried to
rinse them in 70%, 50%, and then 30% ethanol before dissolving in TE or
directly dissolve in TE. They did not resolve at all after a few days.
I noticed that no proteinase was involved in the protocol and it was possible
that the DNA-bound proteins were not removed and wrapped the DNA fabre. Those
protein coulb be denatured by ethanol and other reagents and become insoluble
so that the DNA inside had no chance to be contacted by the water.
I tried to treat the white fabres with proteinase K. It did help. Since then I
turned to other protocol involving proteinase K treatment and got genomic DNA
digestible and PCRable, and good yield, of course.
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