I have been doing two hybrid screening with a whole length protein.
I had go some His+ colonies and then used them for LacZ sceening.
Positives from this screen were then checked using PCR with primers on
either sides of MCS of the 'prey' plasmid. But In all of them I am
getting a 200 basepair fragment- none of the His+, LacZ positive strains
are showing an insert in this PCR screen. I would like to know if
anybody has experienced this kind of problem in yeast two hybrid
sceening. I am using L-40 strain of yeast. As bait plasmid I have
pBTM116 and the library is in pACT2. I really hope somebody can help me
subhash at ccmb.ap.nic.in
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