Ad ITRs and Recombination

[PANGELETTI at bmg.bhs.uab.edu]

		Hi, just thought I'd add in a few thoughts on the Ad ITR recombination 
problem that Graham described.  I noticed this sort of problem when 
tried to put Ad 12 ITRs in a plasmid to construct a minichromosome.  It 
was a very unstable plasmid.  My construct never grew very well.  The 
thing that I would suggest though is using rec- cells to propagate the 
DNA. The growing temp. may also influence recomb. If that doesn't work 
you may be limited to 5 ml cultures.  Certainly, I would be careful 
about subculturing the clone.  Let us know how it turns out.   

Original post:  
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I have prepared a plasmid based vector that contains a large terminal 
fragment of the adenovirus-2 genome.  The vector is 22 kbp and has the 
odd and frustrating ability to recombine and rearrange when grown in 
bacteria in large culture volumes (greater than 5 ml!!)  When I grow the 
bacteria (Dh5 or10) in 5 ml cultures I get no rearrangment but as soon 
as I try to grow a larger culture volume I end up with a ladder of 
plasmids or a new one all together!!!
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Cheers.

Peter C. Angeletti
Department of Biochemistry and Molecular Genetics
University of Alabama at Birmingham
Adenovirus interactions with host cells
PAngeletti at BMG.BHS.UAB.EDU
or 
zm00016 at uabdpo.uab.dpo.edu