confirming yeast 2-hybrid positives / X-gal assay

Weiwei Liu wl126646 at bcm.tmc.edu
Mon Mar 26 11:35:16 EST 2001


Hi, could anyone please tell me the way you usually use to confirm your 
positives resulting from a two-hybrid screen? I mean, when you have too many 
positives to send everything for sequencing, how do you eliminate 
false-positives?

I extracted plasmids from my positives and transformed them back into the bait 
strain, but when I assayed the back-transformants, none of my 22 positive 
clones yielded blue color in the X-gal filter lift assay. Does this mean that 
they're all false-positives? (Oh, I hope not!)

And, if there's something wrong with the X-gal assay, what is the most 
possible reason for it?

Thanks very much for your help!

Weiwei Liu

Department of Biochemistry
Baylor College of Medicine
One Baylor Plaza, Houston, TX 77030

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