Dwindling Plasmid yield *HELP*

[David L. Haviland, Ph.D.]

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).

Martin:

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 
lysed bacteria.

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
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