PJ694A in a two hybrid screen

David Keszenman-Pereyra D.K-Pereyra at sheffield.ac.uk
Wed Mar 1 04:16:33 EST 2000


"does anyone know, when your are using PJ694A for a two hybrid, how can I
determine true positiv in the best way further? I did two hybrid  screen
with a good transformation efficiency. After selection on -His omission
plates I got 400 positives clones. I replica plate this to -Ade an I got
several clones with different color and size. Some are red and others be
white to brown. What is now the best way to determin ethe true positives.
One more question, have anyone a very good positiv control plasmid but p53
largeT? Thank for answering
Best reguards
Richard"

If the bait plasmid is URA3 and the genomic library is LEU2:

Select  for transformants on -UL (-ura and -leucine)
Replica plate on -ULA (-UL and -adenine) and/or -ULH-3AT (-UL and -histidine plus 3-
amino 1,2,4 trizole)
Replica plate positive clones (grown on -ULA and -ULH+3AT) on -UL plus alpha GAL (5-
bromo-4chloro-3-indolyl-alpha-D-galactopyranoside)(either spread 100 ul of 2 mg/ml 
alpha GAL over the entire surface of a  -UL plate using a glass spreader or add 1 ml of 
20 mg/ml to a 1000 ml -UL agar medium at 55 C and pour plates).

Postive candidates are blue and grown on -ULA and -ULH+3AT

Replica plate on FOA-L (FOA plates minus leucine)
Replica plate on -L
                              -UL
                              -LA
                              -LH

Positive clones are white on -L and cannot grown on -UL, -LA and -LH



Good luck !!!!

David Keszenman-Pereyra 
University of Sheffield
Department of Molecular Biology and Biotechnology, D38
Firth Court, Western Bank, Sheffield S10 2TN
UK
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