why does maxiprep always fail for this plasmid??

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3 total messages Started by Mike Levin Tue, 21 Aug 2001 02:07

#18

Author: Mike Levin
Date: Tue, 21 Aug 2001 02:07

18 lines
2211 bytes

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@forsyth.org. Thanks!!

Mike Levin

Re: why does maxiprep always fail for this plasmid??

#19

Author: Emir Khatipov
Date: Tue, 21 Aug 2001 04:19

39 lines
2885 bytes

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@mediaone.net> wrote in message
news:B7A73A9C.1056%mlevin77@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@forsyth.org. Thanks!!
>
> Mike Levin
>
>
>

Re: why does maxiprep always fail for this plasmid??

#17

Author: Chris Jones
Date: Thu, 23 Aug 2001 01:35

16 lines
2011 bytes

In article <B7A73A9C.1056%mlevin77@mediaone.net>,
 Mike Levin <mlevin77@mediaone.net> wrote:

>    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@forsyth.org. Thanks!!

That's happened to me on occasion: once it was clearly a contaminant of 
some sort (very un-Coli odor), but other than that I have no reasonable 
explanation. (Integration into the chromosomal DNA???)

If you can, just get another sample from your original source.

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