Contaminating plasmid

ashendel at aclcb.purdue.edu ashendel at aclcb.purdue.edu
Sun Jan 16 14:58:36 EST 1994


I have lost the original post on this thread but have read all the replies 
posted.

On 15 Jan 1994 18:38:14 GMT, 
Dave Harry  <deh at s27w007.pswfs.gov> wrote:

>>I have absolutely no idea what it is, but I've had the exact same thing
>>happen, TWICE.  I have only seen it with pBluescript, a plasmid of the
>                                          ^^^^^^^^^^^
>Ditto my experience.

Others have seen it with pUC as well. We have seen it with both and pGEM 
also. 

>>size you describe which doesn't cut with the appropriate enzymes.  I even
>                         ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
>Again, ditto.

Does this mean you isolate it and it won't cut (hence either foreign 
plasmid or denatured/ss DNA)?  Or do you mean that you see it remain 
after digesting the plasmid to smaller fragments?

3)Reappearance of this unusual plasmid has occurred only when I use
>>  Xl1-blue cells. I had reordered new plasmid from the company, it was fine
>   ^^^^^^^^^^^^^^
>Again, ditto.
>
>>  new stock and grew in HB101s instead, quite some time later, there is
>>  still no problem.
>
>Nearly ditto.  I used DH5-alpha.
We use DH5-alpha and see it in them.

>(clip)
>
>In short, I never did learn what it was.  *BUT*, the problem did indeed
>disappear after transforming my Bluescript vectors into different a
>different host (DH5-alpha).  (clip)
>
>Until I saw this discussion, I thought we were the only ones who'd
>encountered this problem.  Thanks to all for sharing!!

I wondered also.
>
>I forgot to mention one more thing.  The size of this "mystery
>band" varied with the size of the insert cloned into the Bluescript
>vector.
  
Aha!!!!  A clue.

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.  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



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