2-hybrid system problem (Clontech).

Marieke R. Koedood Zhao rkoedood at bio.bu.edu
Tue Mar 11 13:05:56 EST 1997


Don't know the answer to your problem, but here is some of my experiences.
My original library was in pACT but for the sake of convenient restriction
sites I subcloned the cDNA bits that I got to pACT2. And got just trouble -
a lot of restriction digests didn't make sense at all. I didn find out that
the sequence I got from the WWW was not entirely correct, but that still
didn't explain some of the weird results I got. Someone else then mentioned
that some of these vectors tend to re-arrange, and he found that he should 
avoid letting his bacteria overgrow when making maxipreps of the plasmids.

I finally escaped my problems by going back to my original clones in the
pACT vector (that is NOT pACT2).

Of course you problem may be because of slight differences in linker
sequence that translate into significant differences in conformation of
either your bait or prey-plasmids.

Good luck

Marieke


PC-BS-09 (jr at dna.bio.warwick.ac.uk) wrote:
: 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



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