why does maxiprep always fail for this plasmid??

Tom Vink eikel at xs4all.nl
Tue Aug 21 03:29:23 EST 2001


Of course when it is low copy number (or maybe toxic ?) You have toi
give more details about the plasmid , promotor , origin insert size
etc . If it is low copy number grow more in terrific broth or do an
amplification with chloramphenicol. If your insert is expressed and
toxic you may may trie to repress the expression . (for a lac promotor
add 1% glucose) Hope this helps. Anyhow it is allways nice to know
with what you are working.

Greetings Tom
On Tue, 21 Aug 2001 02:07:17 GMT, Mike Levin <mlevin77 at mediaone.net>
wrote:

>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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