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
