Q : E. coli instability

"Alexander Kraev" bckraev at aeolus.ethz.ch bckraev at wawona
Tue Mar 14 05:59:01 EST 1995


In article <Pine.SOL.3.91.950312232409.23202B-100000 at dale>, Elizabeth Lawrence <ez055325 at peseta.ucdavis.edu> writes:
>Hi fellow cloners,
>
>I am having problems with JM109 E. coli maintaining my plasmid.  The 
>plasmid is a vector plus a viral genome from which I have deleted about 
>800bp.  I have gotten colonies from electroporation of these bacteria and 
>up to this point I know I have the right DNA fragment-from gel analysis.  
>However, after culturing and maxi-prepping it appears that only the 
>vector was maintained and the entire viral genome was "kicked out".  
>Please let me know if you have a clue as to why this may be occurring.
>
>Thanks in advance, Liz  eclawrence at ucdavis.edu

Can it be that your initial clone was not purified from a neighbouring colony?
If so, the latter overgrow yours in large-volume culture, especially if your clone
has an abnormally long generation time ( which is not unusual ).  However, if
your clone is truly unstable, it may help to return to your original miniprep
DNA, transform it back into the same ( or other ) strain and plate to get well
separated colonies. If you then grow many separate small cultures, isolate DNA
and check it on a gel, AND ONLY THEN POOL the right sized clones, you may get
out of this situation. Good luck


*************************************************************************
Alexander Kraev, Ph.D.                 Internet: bckraev at aeolus.ethz.ch
Lab. of Biochemistry III               Phone: 0041-1-632-31-47
Swiss Federal Institute of Technology  FAX:   0041-1-632-12-13
Universitaetstr.16
CH-8092 Zurich
"Some ideas are obscure not because they are complex, but because they 
 are excluded from our circle of comprehension" - Kozma Prutkov



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