why does maxiprep always fail for this plasmid??

Nick Theodorakis nicholas_theodorakis at urmc.rochester.edu
Tue Aug 21 09:44:43 EST 2001


In <B7A73A9C.1056%mlevin77 at mediaone.net>, Mike Levin 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!!

Sounds like you got an older low-copy plasmid (e.g., one based on 
pBR322). Do you know what vector was used to construct it?

Nick

-- 
Nick Theodorakis
nicholas_theodorakis at urmc.rochester.edu

 




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