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Plasmid Preps from MRSA

Plasmid Preps from MRSA - Protocols and Methods Forum

Plasmid Preps from MRSA - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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Old 07-09-2004, 11:38 AM
Anthony C. Hilton
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Default Plasmid Preps from MRSA



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


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Old 07-10-2004, 12:04 AM
Jose de las Heras
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Default Plasmid Preps from MRSA


"Anthony C. Hilton" <[Only registered users see links. ].uk> wrote in message
news:ccm048$6l2$[Only registered users see links. ].uk...
to
DNA
to

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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