Joseph C. Bagshaw
jbagshaw at wpi.edu
Sat Jun 6 16:29:23 EST 1998
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
> 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
> 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
> 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
> 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
> Brigham and Women's Hospital
> Boston,MA 02115
> email: jaugliera at rics.bwh.harvard.edu
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