undigestable genomic DNA

Hiranya S. Roychowdhury hroychow at nmsu.edu
Wed Feb 16 11:00:54 EST 2000

Genomic DNA samples need to be digested for extended periods. I do not think
that a 2h digestion is enough, unless you are using very high amts of
enzymes. Usually we carry out the digestion at least 24h. Sometimes we spike
the reaction with the RE and let it go for another 12-18h. That is because
for a Sounthern analysis of a target that is not highly repetitive, one
needs at least 10ug of digested DNA per lane. So, a large amount of DNA
needs to be digested to begin with. 
Including spermidine at a final conc. of 4mM in the reaction may help those
difficult to digest samples.

At 03:30 PM 2/16/00 +0100, Gregor Bucher wrote:
>Hi southern-freaks!
>I want to do a southern with genomic DNA (insect). When I digest the DNA
>with Hind3 it looks fine. With EcoR1 and Xho however, there is quiete a
>bit high molecular DNA that obviously has not been digested (digesting 2
>hours or o/n does not change things). Does anybody have experience with
>genomic southerns?
>Should I proceed or start with a new preparation of DNA? 
>Thanks for comments!
>Gregor Bucher, Zoologisches Institut der LMU München
>st2153 at zi.biologie.uni-muenchen.de

Dr. Hiranya Sankar Roychowdhury
New Mexico State University
Las Cruces, NM 88003
Ph. (505) 646-5785
hroychow at nmsu.edu


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