HELP! Genomic DNA not cutting!

John Ladasky 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:
>Hello!
>
>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.
>
>Many thanks,
>Mary
>

Greetings, Mary,

	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



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