DNAse contamination in plasmid preps

George Theodoris ez003450 at bullwinkle.ucdavis.edu
Thu Nov 10 19:57:04 EST 1994


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?



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