2-hybrid system problem (Clontech).

[PC-BS-09]

Dear all.

	Could someone out there describe what's going on with my two hybrid 
system. 

	The ultimate aim is to screen a human lung library (in pACT2 bought from 
Clontech) against a known viral protein. Currently, I have a two viral 
proteins that are known to interact and are in the low expression vectors 
pGAD424 and pGBT9 (Clontech). Using these vectors the proteins interact and 
blue colonies can be observed with the B-gal filter assays. However, once the 
proteins are transferred to the high expression vectors (into their equivalent 
ones), pAS2-1 and pACT2 (Clontech), no interaction is observed i.e no blue 
colonies are observed. I am using the yeast strains Y187 and SFY526 and get 
the same result in both strains.

	Upon pairing the viral gene in the new vector with its counterpart in the old 
vector, still no interaction is observed. I know that the viral gene are in frame with the 
vector ORF's and therefore should be expressing. Why am I not detecting any 
interaction from the constructs in pAS2-1 or pACT2????? 

		The vectors were initally transformed into E. Coli XL1-blue and then 
grown up and preped using the Qiagen Maxi prep kits.


Thanks in advance


Jaz
jr at dna.bio.warwick.ac.uk