Dwindling Plasmid yield *HELP*
David L. Haviland, Ph.D.
HAVILAND at KIDS.WUSTL.EDU
Mon Apr 25 22:04:18 EST 1994
In <leach-200494114012 at med-pharm5.bu.edu> leach at mbcrr.harvard.edu writes:
> 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).
This may not apply to your situation but we have had something like this
before in our lab. The cause turned out to be some sort of phage
contamination of our bugs that contained our eukaryotic expression vector
(with cDNA) of choice. Do you notice any "stringy-stuff" accumulating
inyour cultures with overnight incubation? This would indicate
In our case, our yeild of plasmid kept dropping until we were only getting
a few hundred micrograms from 2 liters of TBroth, and with each successive
plasmid prep the mess of lysed bacteria seemed to increase. It goes
without saying that we had to do successive plasmid preps as didn't get the
yield needed the last time we did it, ergo - we furthered the infection
until the 'sharp-eye'd' tech in the lab looked at the bugs and said "That
looks like a phage infection!" I should note that she's our local expert
in phage preps as well... grin!
In any case, I hope this helps.
+ David L. Haviland, Ph.D. Internet:"haviland at kids.wustl.edu" +
+ Washington Univ. School of Med. A.K.A : The Compiler +
+ Dept. of Peds./Pulm. Box 8116 ICBM-Net : Just hit St. Louis +
+ 400 S. Kingshighway &-6 <- User is Brain Dead... +
+ St. Louis, MO 63110 FAX: 314-454-2476 +
+ (314) 454-6076 +
+ ------------------------------------------------------------------- +
+ Outside of a cat, a book is a man's best friend. Inside of a cat, +
+ it's too dark to read! +
More information about the Methods