Cycloheximide selection in yeast-2-hybrid
Simon.Dawson at nott.ac.uk
Wed Aug 26 02:47:05 EST 1998
In article <35E1B04E.1BDC343D at iib.uam.es>, Miguel Aguilera
<maguilera at iib.uam.es> wrote:
>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!
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.
Dr. Simon Dawson
Laboratory for Intracellular Proteolysis
Molecular and Cellular Biology Section
School of Biomedical Sciences
The Medical School
Queen's Medical Centre
TEL:+44 (0)115 9249924 ex. 44787
FAX:+44 (0)115 9709969
Email:Simon.Dawson at nott.ac.uk
"Back off man, I'm a scientist!" - Bill Murray.
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