Loosing expression efficency

Tobias Straub tobias.straub at mail.uni-wuerzburg.de
Mon May 3 10:29:09 EST 1999


In article <37285CFE.D3F924BE at imiucca.csi.unimi.it>, One Night 
<molla-remove- at imiucca.csi.unimi.it> wrote:


> Hello everyone;
> I have a very great problem:
> I built a new expression vector derived from pET9 eand pT7.7
> and cloned my cDNA for expression. The recombinant plasmid
> was then inserted in the BL21 DE3 pLysS strain.
> The first exp. test was very good (we can consider it as the 100% of
> the exp. level ). I had not success in reproducing this result because:
> the exp. level is very variable (if I make a plate from my glycerol
> stock
> and pick 4 colony from it I obtain 4 different exp. levels (let's say
> 25, 31
> 55, 60% in respect of the original). The second thing is that the
> overall
> exp level seem to decrese from the beginig of the test (now I have only
> about 15%).
> Anyone has an idea to explain these facts?
> Note:
> I tried everything like inducing and collecting at different times,
> making again all the stock solutions, and so on.
> The resistance is Kanamicyn (and Chloramphenicol for pLys cells)
> but I don't think that the losing of plasmid is the problem because I
> start every time from a fresh plate from the glycerol stock and never
> let the cell to grow for a too long time.
> Anyway is there a test to know if the cells had got the Kanamicyn
> resistance and had loose the plasmid?
> Thank you
> dr. Gianluca Molla
>
> molla at imiucca.csi.unimi.it
>

at first: How do you quantify your expression levels?

- my only tip so far: don't use glycerol stocks. never, never.


--

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Tobias Straub
Medizinische Poliklinik, University of Wuerzburg
Klinikstrasse 6-8, 97070 Wuerzburg, Germany
Phone: +49-931-2017085      Fax: +49-931-2017120
Tobias.Straub at mail.uni-wuerzburg.de
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