Problems with dissolving genomic DNA

Philip L. Carl plc at med.unc.edu
Tue Jul 13 15:17:20 EST 1993


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.



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