Contaminating plasmid

James N Petitte jnppo at unity.ncsu.edu
Mon Jan 17 10:45:23 EST 1994


ashendel at aclcb.purdue.edu wrote:

: I have long seen this 2.0 to 3.0 kb band with pUC and and pBS constructs in 
: DH5alhpa cells.  I found that it was always possible to identify by its 
: reduced abundance (by EtBr staining) relative to the expected quantity of 
: a fragment its size and its size (always about 1 to 2 kbp smaller than the 
: largest band on the gel).  This last observation is similar to that in the 
: above post.

: In addition to the observed size dependence, I also have seen that this 
: band ONLY appears on gels of digests of plasmids that are very HEAVILY 
: loaded.  

We first noticed it in large-scale preparations of pBluscript (no insert) 
using the Qiagen columns.  Likewise it would show up when loading large amounts of 
prep and would appear as a faint band.  We also noticed it in the 
"example" gel of pBluscript published by Qiagen.  I don't know why 
Strategen has never heard of this.  We decided to ignore it because it 
was present in such low amounts.

Since we tend to do the same method for minipreps and digest 
: volumes, a heavily loaded gel means a high yielding miniprep.  This tends 
: to occur only with pUC, pGEM, and pBS plasmids in my lab.  I have suspected 
: this is an artifact of the large amount of plamid in the miniprep, occuring 
: during the isolation procedure (alkaline lysis) or during the running of 
: the gel.  Since the size of the band seems to vary with the largest 
: fragment on the gel, not the size of the intact plasmid, and the 
: mystery band disappears with reduced loading of DNA on the gel, my current 
: thought is that it may be a minor structural variant of large pieces of 
: linear DNA that fold in half (or something like this) and migrate in the 
: gel more rapidly than expected for their size.  I know this sounds odd, but 
: it fits my observations.  It also is consistent with the lack of any 
: isolation of a foreign plsmid and with the tendency to occur with ultra 
: high copy number plasmids and with  XL-blue vs DH5alpha or with DH5alpha vs 
: HB101, i.e.,cell lines that yield higher amounts of plasmid.  

: I've since not worried about it, but it often is worrisome to new students. 

: Comments?
: Curt Ashendel
: Purdue University
: West Lafayette, IN
: ashendel at aclcb.purdue.edu

James Petitte
NCSU
Raleigh, NC




More information about the Methods mailing list