cDNA library in pACT-vector - (Jan/10/2006 )
Hello everybody!
I am having a problem with a 2-hybrid screen i did last week 
! I got two putative interaction targets and want to confirm the result now by doing a 2-hybrid check with the bait and the two candidates only. The problem is that the library was delivered as a lambda-library, therefore I do not have the vector with the activation domain (pACT) alone, but only linked with the cDNA-library.
I know that I can buy a newer version of the AD-vector from clontech, but they use a new system that is not compatible to mine (and I do not want to start screening all over again!).
The cDNAs are inserted at a XhoI restriction site.
My plan is to transform coli´s with pACT-cDNA library (ca. 500 ng), pick individual clones (I do that to limit the amount of cDNA-pACT to hopefully 1 vector with one cDNA-insert per colony), then digest the vector with XhoI, cut the band with the appropriate size (linearized vector without cDNA =approx. 8kb) out of the gel, and religate the vector.
If I am correct, I then have the empty vector, which can be used for further cloning. Do you agree? Do you think that this is a good plan? Does anybody have experience with cutting an insert out of a vector, religating it and using it again with another insert? Or is there a simpler way to get the empty vector (except buying it, because you can´t)? 
Thank you for your help in advance!
Ronnie
I think it's no necessary to make the empty vector.
You can extract plasmid DNA from yeast that contain the bait and your candidate vector,thansform to the e.coli. plate on LB medium containing the antibiotic markers in AD vector.Then extract plasmid from the E.coli.,that's the ad vector with your candidate.You can confirm the result by transform your bait and the plasmid to yeast...
I am having a problem with a 2-hybrid screen i did last week
! I got two putative interaction targets and want to confirm the result now by doing a 2-hybrid check with the bait and the two candidates only. The problem is that the library was delivered as a lambda-library, therefore I do not have the vector with the activation domain (pACT) alone, but only linked with the cDNA-library. I know that I can buy a newer version of the AD-vector from clontech, but they use a new system that is not compatible to mine (and I do not want to start screening all over again!).
The cDNAs are inserted at a XhoI restriction site.
My plan is to transform coli´s with pACT-cDNA library (ca. 500 ng), pick individual clones (I do that to limit the amount of cDNA-pACT to hopefully 1 vector with one cDNA-insert per colony), then digest the vector with XhoI, cut the band with the appropriate size (linearized vector without cDNA =approx. 8kb) out of the gel, and religate the vector.
If I am correct, I then have the empty vector, which can be used for further cloning. Do you agree? Do you think that this is a good plan? Does anybody have experience with cutting an insert out of a vector, religating it and using it again with another insert? Or is there a simpler way to get the empty vector (except buying it, because you can´t)?
Thank you for your help in advance!
Ronnie
No, I think you should be running a negative control by testing the 2 pACT clones you pulled out against the empty BAIT vector ie. the one with the DNA-binding domain.
The key thing you need to identify is: Do the two pACT clones generate reporter gene expression autonomously (ie. when co-transformed with DNA-binding domain only) or do they require your bait construct (ie. DNA-binding domain fused to protein of interest). The former case = bad, the latter case = good.
The negative control you are planning on doing is less useful, because if your bait was an autonomous activator, surely you would have pulled out thousands of +ve clones. So you probably don't need to worry about your bait.