Rearranging Plasmids

[Carrington]

The type of rearrangment problem described with Ad-2 DNA-containing 
plasmids is also seen when we grow plasmids that contain full-length cDNA 
representing the tobacco etch virus genome (plasmid=14 kb).  We see 
consistent rearrangements when cultures larger than 5 ml are grown.  

Our solution...we do not grow cultures with volumes larger than 5 ml.  
Problem solved.  When we need large-scale plasmid preps, we grow 40 or 80 
5ml cultures for ~20 hrs in large Pyrex culture tubes.  They are then 
pooled and extracted.  Prior to growing the liquid cultures, it is 
important to streak your colonies on agar plates and only pick only the 
"small" colonies.  The large colonies may have deletions and 
rearrangments.

One final comment for those with the patience to read this far.  It is 
nice to see postings that discuss technical problems, research methods, 
virological research issues, recent unexpected findings, etc., at this 
site.  These are the contributions that most benefit the scientific 
community.  Perhaps we could see more of a focus on this type of 
discussion rather than time-consuming re-hashing of the meaning of life 
and whether or not viruses are alive. 

Jim

James C. Carrington
Department of Biology
Texas A&M University
College Station, TX  77843
Phone: 409-845-2325
Fax: 409-845-2891
e-mail: carrington at bio.tamu.edu