Plasmid Preps from MRSA

Jose de las Heras jose.delasheras at virgin.net
Fri Jul 9 19:04:04 EST 2004


"Anthony C. Hilton" <a.c.hilton at aston.ac.uk> wrote in message
news:ccm048$6l2$1 at wotsit.aston.ac.uk...
> Hi
>
> We have been trying to isolate plasmid DNA from clinical isolates of MRSA.
> In some cases we find plasmid (as expected?) and in others we are unable
to
> isolate plasmid DNA.  One possibility is that there is indeed no plasmid
DNA
> present, alternatively it might be that we are not isolating plasmid due
to
> difficulties in lysing the cells.  We are running positive controls from
> which we obtain plasmid in each case but were wondering if anyone might
> suggest a method we could use to confirm the cell lysis and plasmid DNA
> precipitation step.
>
> Thanks in advance
>
> Anthony Hilton
> Jon Caddick

I'm sure there will be more elegant ways, but the first thing that came to
mind was the following: while doing a normal plasmid prep, we avoid lysing
for too long and we are relatively gentle during that step, to avoid getting
chromosomal DNA with our plasmid DNA. So, if you lyse for longer than usual
and vortex, you're bound to get chromosomal DNA, which you can easily test
for. If you get chromosomal DNA, the bacteria must have lysed... if you
can't detect any plasmid, then there's probably none. Does this make any
sense to you?

Jose





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