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2-hybrid - plasmid rescue

B.E. Causier BGYBEC at leeds.ac.uk
Mon Jul 21 03:51:11 EST 1997


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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