pGBKT7 Yeast two hybrid - Problems while using pGBKT7 (Feb/04/2006 )
I´m using pGBKT7 as a bait-vektor in an Yeast-two-hybrid screen. I found about 200 positiv clones. After sequenzing i recognized that 90% of these clones carrying the transaldolase-gen (as the prey).
The problem now ist, that the transaldolase has just nothing to do with my bait-gen, maybe the reportergenes are expressed because the transaldolase-gen interacts with pGBKT7 and not with my bait.
Can anybody help me, or does anybody now something about this problem?
I`m sorry, cause my english isn`t that good, i hope you can understand what i`m talking about.
Thanks a lot
Are you screening your library by mating or co-transformation?
I am guessing you are mating your bait-containing cells to cells containing your library of interest!
I have used both methods and although the mating protocol has its advantages I have found that I do get alot of the same clone (or same few clones). And they are not necessarily the same clones that I pull in independant mating screens! My AH109 library is good, and representative.
My current explanation (for lack of a better one) is that early on a few mated cells get propagated (maybe because they allow the cell to grow better?). In my case my bait is slightly posionous, so any false positive which would aleviate this might explain why this is occuring.
My suggestion to you would be to try co-transformation w/your bait and ds-DNA library. While this does not eliminate the many false positives you are likely to have to wade through, at least they are different clones and better represent the library you are screening!
Or you could try screening independent matings (using the same library strain) to see if the prey is different each time.