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
> Good luck,
> 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
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
I really want to hear a real story.
Thank you so much for your comment again!
More information about the Methods