Plasmid Problems!!!!

[Joseph C. Bagshaw]

Sounds like you've got a yeast infection.

********************  HAVE GENES, WILL TRAVEL  ********************
Joe Bagshaw, Worcester Polytechnic Institute
jbagshaw at wpi.edu
Roadkill on the information superhighway.

On 29 May 1998, John Augliera wrote:

> Involved in a positional cloning project and having problems with my plasmid
> preps.
> 
>  Some background:  1. The cloning vector is pBluescript
> 		   2. The bacterial strain is DH5alpha
> 		   3. Using the ABI 377 automated sequencer
> 
> The problem started about a month ago.  We were initally doing the plasmid
> preps using Qiagen's spin column prep kit.  The sequencing was working fine
> with only the occasional clone failing to sequence.  Routinely about 2 out of
> 36 sequencing rxns would fail but could usually be explained by a lower than
> expected yield from the plasmid prep.  We started to gear up for high
> throughput
> sequencing and needed to prep a large amount of clones quickly and cleanly.
> The solution was Qiagen's 96-well Turbo prep kit.  It uses a vacuum manifold
> for clearing the lysate and eluting of the DNA.  I started to see a problem
> immediately with the sequencing of the clones.  The failure percentage on the
> sequencing started to rise to 50% or more of the rxns not working.  I ran the
> clones of the failed sequencing rxns out on agarose and saw no band.  We tried
> repreping the failed clones with the tried and true spin column kit but still
> no plasmid.  This seemed very strange because the clone  prior to the prep
> had been cultivated on LB/ampicillian plates and grown in liquid culture with
> antibiotic selection.  How could the cells grow without plasmid????
> 		One of our lab people suggested the problem maybe
> incomplete lysing and tried incubating the the resuspended cell pellet in
> lysozyme before
> doing the Qiagen prep procedure.  Amazingly this seemed to help and we
> saw bands on the agarose gel but not all of the failed preps produced plasmid.
> Which still leaves me wondering how the cells are growing in the selective
> media???   Others in the lab have noticed failed preps before but apparently
> they paid it no mind since the problem occured few and far between in
> frequency.
> I asked if they had used the same competent cells and some of them had so
> it got
> me to wondering if something might be going on.  The cells were made
> competent by a technician last February.  I thought competent cells were
> good for about a
> year at -70 in glycerol media.  Since the post-doc that did the transformations
> saw no problems with the number and ratio of blue/white colonies on her plates
> she assumed the cells were fine and continued to use them.   I recently
> streaked
> out some of the competent cells on AMP+ and AMP- plates.  As expected nothing
> grew on the AMP+ plates.  The AMP- plates seemed to show more than just one
> colony type on the plate.  The usually opaque colonies of the DH5 strain
> were interspersed with intensely white colonies.  If this is a contaminating
> strain could this be cause of our headaches????
> 
> 
> Any thoughts or suggestions are greatly appreciated!!!!
> 
> John Augliera
> Technician
> Brigham and Women's Hospital
> Boston,MA 02115
> 
> email: jaugliera at rics.bwh.harvard.edu
> 
> 
> 
>