why does maxiprep always fail for this plasmid??

Emir Khatipov khatipo at ZAPuchicago.edu
Mon Aug 20 23:19:41 EST 2001


It is quite probable that your plasmid has a very low copy number. You
should be able to find that out from the person you got it from or by
checking the type of plasmid. Try to grow more cells than recommended by
Qiagen. The latter may result in a less pure DNA, and thus you may want to
re-precipitate DNA with ethanol or isopropanol a couple of times after
Qiagen procedure.

- Emir


"Mike Levin" <mlevin77 at mediaone.net> wrote in message
news:B7A73A9C.1056%mlevin77 at mediaone.net...
> Hi All -
>
>    I'm hoping you can shed light on the following phenomenon. In my lab,
we
> often get plasmids in the mail. To use the construct, we transform them
into
> E. Coli (JM109 competent cells from Promega, mostly), grow them up in the
> presence of an antibiotic (Ampicillin usually), and then do Quiagen
> maxipreps to get the DNA. Usually it works fine - no problem. However,
there
> is one recent plasmid we got which just won't prep up! It transformed fine
> onto an Amp plate, grew up fine in 300 ml of overnight shaking culture of
LB
> + Amp, and when spun down made a bacterial pellet of the same size as
others
> done that day. However, when the Quiagen prep was done, the yield was very
> low (< 0.1 mg/ml). The other 5 preps done that way worked fine. This
> situation was repeated 3 times over the last month, and this one plasmid
> just won't work! What could possibly account the fact that the bacteria
grow
> fine, but I can't get the plasmid prep out?? Any ideas would be greatly
> appreciated - please email to mlevin at forsyth.org. Thanks!!
>
> Mike Levin
>
>
>





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