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.