Dwindling Plasmid yield *HELP*

Steven Coon at umich.edu
Fri Apr 22 10:00:42 EST 1994


 
> In the promoter-luciferase constructs (no SV40 enhancers) i am finding
> several constructs have very little yield after maxiprepping. I use the
> Qiagen tip method - (i know it is not the columns or solutions as other
> plasmid preps come out fine while some consistently come out crap!
> 
> I have tried re-transforming the bad plasmids into bacteria, but so far no
> joy. Someone else in the lab is having a problem with this
> promoter-luciferase construct also (different promoter though).
 
> 
> p.s. I am gonna try terrific broth vs. Luria Broth (we usually use this), i
> am also going to increase ampicillin from 25ug/ml to 100ug/ml.
 

 	Increasing the amp will only decrease your yeild even more. I would
measure the amount of bacteria you are getting. Is it the same as other
preps? If your inserts are longer in these preps your yeild will be less
than those plasmids which have shorter inserts. You could try Cscl
centrifugation and continue to  use more bacteria. You may consider using
chloramphenicol to amplify your plasmid. 


Steve Coon
sdcoon at umich.edu
University of Michigan Medical Center



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