low plasmid yield

Tom Thatcher ttha at troi.cc.rochester.edu
Wed Nov 24 10:33:49 EST 1993


In <57491 at sdcc12.ucsd.edu> wsun at jeeves.ucsd.edu (Fiberman) writes:

>This is for all the microbiologists:

>I have been working with a clone in Bluescript SK.  I modified
>the vector a little by deleting some regions of the multiple
>cloning site.  Now this modified plasmid, when used to transform
>XLI blue, makes them grow very slow.  Moreover, when I try to
>extract this plasmid using a boiling miniprep procedure, I get
>very, very low yields.  It seems like the bacteria is more
>resistant to lysis.  Do you have any idea why deleting some of
>the MCS could result in these phenomenon?  Could it be that the
>bacteria is expressing the clone somehow?

>-fm

BLuescript generally has an expression problem in lacZ hosts--
we have had problems with a number of different inserts that
we believe are due to incomplete repression of transcription.
It is unlikely that merely deleting the mcs would have an affect,
since most double enzyme cloning deletes the inbetween
sites anyway.  You might try a non-expressing vector.

Tom Thatcher
University of Rochester
--
Tom Thatcher
University of Rochester
School of Medicine and Dentistry
ttha at troi.cc.rochester.edu 



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