low plasmid yield

nishir at ohsu.edu nishir at ohsu.edu
Wed Nov 24 17:34:28 EST 1993


In article <1993Nov24.153349.23507 at galileo.cc.rochester.edu>
ttha at troi.cc.rochester.edu (Tom Thatcher) writes:
>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 
>
But we almost always use XL-1 blue as a host for Bluescript and have never seen
this problem of slowed growth.  In fact, our plasmid yields are usually
excellent- 100-200 ug per 30 ml culture in TB... What else could be going on?

Rae Nishi
OHSU
Portland OR



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