Cycloheximide selection in yeast-2-hybrid

Simon Dawson Simon.Dawson at nott.ac.uk
Wed Aug 26 02:47:05 EST 1998


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In article <35E1B04E.1BDC343D at iib.uam.es>, Miguel Aguilera
<maguilera at iib.uam.es> wrote:


>Hello everybody!
>
>Did anybody use the cycloheximide dominant selection marker in pAS2-1 of
>the Matchmaker-2 kit? Does it work in the first generation or several
>passes are needed to recover the colonies that lost this plasmid?
>
>Any comment is welcome! 
>

Hi Miguel,
        Yep. I have recently used the CHX marker for rescue of prey plasmids
in a two-hybrid screen. I have to admit that my impression was mixed. I
tried it on 9 clones of which maybe 5-6 gave me colonies with strong growth
on CHX plates (I always got a strong background growth with the Y190 cells I
was using). Some of the clones never gave any resistant colonies but I was
able to cure the Leu+Trp+ strain of the bait plasmid by culturing the strain
in media containing Trp for a few days and then plating out.
    Of the 5-6 clones that gave me CHX resistant colonies, some wouldn't
give me anything unless I grew the Leu+Trp+ parent strains in Trp+ media foe
a few days before plating onto CHX media.
    Overall, it seemed like a great way of curing the bait plasmid out but
wasn't as effective as they suggested it would be......surprise, surprise!
    Hope thats of some use to you. All the best.


            Simon.

--
Dr. Simon Dawson
Laboratory for Intracellular Proteolysis
Molecular and Cellular Biology Section
School of Biomedical Sciences
The Medical School
Queen's Medical Centre
Clifton Boulevard
Nottingham
NG7 2UH
U.K.

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