HELP! Genomic DNA not cutting!
ladasky at leland.Stanford.EDU
Thu May 11 22:07:37 EST 1995
In article <1995May11.191903.7840 at mcrcr6>,
<galinm01 at mcrcr6.med.nyu.edu> wrote:
>Seeking suggestions on "how to" "clean-up" genomic DNA that
>refuses to digest with restriction enzymes. Repeated phenol/chloroform
>extractions have not helped.
>This DNA sample is semi-precious. Will be grateful for advice and comments.
>This problem has not been encountered before in this lab.
>Numerous other preps of genomic DNA have been made with the labs
>standard isolation procedure, and have digested just fine.
I'm dealing with almost the same problem at the moment. I have
some genomic DNA preps that I *was* able to cut a year ago, but now I can't.
And I'm using the same restriction enzymes and suppliers. I had a look at
my DNA in some ethidium bromide on a sequencing plate atop a UV transillum-
inator. I had tiny "clumps" which would not disaggregate when passed through
a pipet. Very little of that DNA would even run into the gel. I just
finished a repeat phenol/choloroform/isoamyl alcohol extraction on this DNA,
and as part of the final resuspension I placed the DNA on a Thermo-mixer at
65 degrees for an hour. This seems to have helped - now most of the DNA
runs onto the gel but is clearly larger than 23 kB. Right before I run the
restriction digests, I'll repeat the 65-degree step after diluting the DNA
into 1x enzyme buffer.
Hope this helps. Anyone interested in my results, or who has advice
to offer, can send me email.
Unique ID : Ladasky, John Joseph Jr.
Title : BA Biochemistry, U.C. Berkeley, 1989 (Ph.D. perhaps 1998???)
Location : Stanford University, Dept. of Structural Biology, Fairchild D-105
Keywords : immunology, music, running, Green
More information about the Methods