PBR322 - maximizing yields
bchs1b at Elroy.UH.EDU
Thu Sep 3 09:56:14 EST 1992
In article <email@example.com>, bhjelle at carina.unm.edu writes:
>In article <3778 at fcs280s.ncifcrf.gov> pnh at fcs260c2.ncifcrf.gov (Paul N Hengen) writes:
>>>bhjelle at carina.unm.edu () writes:
>>:Unfortunately, some things can only be done in pBR-based
>>:vectors and I have been plagued with totally miserable
>>:yields of one particular example of same for more than
>>:a month now.
>>I have two thoughts about your problem...
>>1. You may have cloned a segment of DNA that would prohibit
>>high copy number in any plasmid, in which case you will be
>>wasting much effort trying to amplify.
>>2. Why not spend some time subcloning into one of the
>>runaway plasmids that can be amplified simply by changing
>>temperature? This could save quite a bit of aggravation
>>and the bottle of chloramphenicol. See these refs...
>>B. E. Uhlin et al. Gene 6:91-106. (1979)
>>B. E. Uhlin et al. Gene 22:255-265. (1983)
>Actually the plasmid is a trpE expression construct, and I
>am having the most problems with the vector itself, rather than
>derivitives containing my inserts. The lab I got the clone from
>swear that it grows about as well as pBR, and that there is
>enough tryptophan in LB to repress the trp E anyway.
>I could move the whole trpE gene into pUC I suppose, but that
>would require being able to grow the pBR-based vector.
Amplification should certainly help. But another suggestion is to try
a different E.coli strain. I certainly find that I get higher plasmid
yields in recA+ strains than in recA-, so for example if you are in
HB101 or DH series you might want to try something else.
Have you had trouble getting good yields with other plasmids. The problem
may be something other than this plasmid, just that with pUC vectors even
a bad yield still gives plenty of DNA. You might look over your plasmid
prep reagents etc.
Department of Biochemical and Biophysical Sciences
University of Houston
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