Deletion of plasmid

Jung Hoon Ahn jhahn at bioneer.kaist.ac.kr
Wed Jul 15 20:52:51 EST 1998


Bernard Murray wrote:

> I can echo Duncan's advice as I also had problems with an ABC
> protein.  Make sure that the F' (and thus the Iq) is present
> in the XL1-Blue (I think this requires tet selection)
> and do not use blue/white selection with the construct
> (you can screen by PCR or restriction digestion of minipreps).
> Both these conditions should reduce leaky expression from        
> the Lac promoter.  For a handy non-Lac plasmid try one of Promega's
> old pSP plasmids (eg. pSP64).  If you think the bugs are part of the
> problem then maybe try HB101, SURE, TOPP etc.  You could also
> try changing the culture conditions (richer broth, lower temperature
> etc.).
>      Good luck,
>           Bernard
> --
> Bernard Murray, PhD
> Dept. Cell. Mol. Pharmacol., UCSF, San Francisco, USA
 
Thank you so much for your great advices.
I've performed transformation on the McConkey plate without tet,
which may allow leacky expression of lac promoter.
I might have found a plasmid without deletion doing like that.
 
If I succeed in getting the plasmid, can I express it?
Based on your suggestion, I can grow the cell in LB broth containing tet
at low temperature (25 oC) to minimize the expression of ABC
transporter,
then can I express it by adding IPTG.
Would it give E.coli chance to change the plasmid?
 
Could I hear your experience with your ABC protein.
How did you overcome the difficulties with expression ABC
transporter.    
I really want to hear a real story.
 
Thank you so much for your comment again!



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