DNAse contamination in plasmid preps

[Michael Cooley]

George Theodoris (ez003450 at bullwinkle.ucdavis.edu) wrote:
: Has anyone else had a problem with degradation of plasmid preps?
: When I first started in my current lab I was having a terrible problem 
: with having my plasmids preps degrade in an unusual way. When the plasmid 
: degraded it ran at the same length as RNA runs so at first I thought the 
: RNAse I was using was not working. 
: I found that several steps eliminated the problem:
: 1) Growing my Bacteria in LB rather than TB
: 2) Reducing the growing time to no longer than 14 hours
: 3) Switching from a boiling prep to alkali-lysis
: 4) Resuspending the DNA at a concentration not greater than 
: 1microgram/1microliter
: Now someone else in my lab is having the same problem and I don't know 
: what to tell her since she basicly follows the above steps except that 
: she resuspends her DNA at a higher concentration. My experience was that 
: the more concentrated the DNA was the more it was degraded. Extracting 
: with Phenol-Chloroform did not help, but purifying the DNA on CsCl/EtBr 
: usually did the trick. Has anyone else experienced this strange problem?

I had a similar problem a while back and found that the problem was with 
the genotype of the bacteria. If the bacteria is HB101 it has the 
wild-type endA gene. I switched to JM109 and the problem went away.


#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%
%                                                        #
#   Dr. Michael Cooley         Practice Random Kindness  %
%   mbcooley at ucdavis.edu    and Senseless Acts of Beauty #
#                                                        %
%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#%#