2-hybrid - plasmid rescue

Marieke Koedood Zhao marieke at bu.edu
Tue Jul 22 13:02:57 EST 1997



I had the same problem. The way I got around it was by streaking and 
re-streaking on -Leu plates (i.e. with Trp), picking colonies and 
testing by replicat plating which colonies have lost the bait (DNA binding
domain fusion) plasmid. Using these colonies for plasmid rescue gave me
the library plasmid.

My guess is that the bait plasmid gets amplified, possibly because of the
3-AT selection.

Good luck

Marieke
'

B.E. Causier (BGYBEC at leeds.ac.uk) wrote:
: Dear yeast netters,

: I'm experiencing a few problems isolating the library plasmid following a two 
: hybrid screen. I'm following the Clontech method for isolating the library 
: plasmid using the HB101 E. coli strain and growing transformants on minimal 
: media minus leucine. However, when I investigate the plasmids prepared from 
: these cells they all turn out to be the DNA-binding domain plasmid!

: I have analysed the plasmids rescued from my Leu+, Trp+, His+, LacZ+ yeast
: strain by restriction digest and cannot see any activation domain plasmid
: on the gel.

: So, my question is - how do I rescue the library plasmid? My only thought was 
: to retransform the rescued plasmids back into yeast, isolate Leu+ colonies 
: and rescue plasmid from these cells. However, if the BD-plasmid is so 
: predominant I'll be lucky if I see any Leu+ colonies.

: If anyone has any suggestions/comments I'd be glad to hear them.

: Cheers,

: Barry.




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