No comment on the lack of specificity of your retransformed plasmids.
But the lower activity is not unusual. In my case I'm pretty sure I have
the real thing based on functional studies in other systems. Recently
retransformed into the 2-hybrid system and found no (!) activity of my
original cDNA (original meaning fused to the GAL4 activation domain).
I have 2 explanations for this, the one more disturbing one us that my
'original' clone is not the real thing any more. This based on some
observations about the instability of the vector.
The other, more relevant to you maybe, and also based on definitve
observations, is that in my Y2H screen I selected for increased copy
numbers of the bait and/or the 'fish'. Therefore the nice positive result.
Upon retransformation I introduce lower copy numbers of both and don't
get the activity I saw before. Testing this hypothesis now by 3AT
selection. But I should add that I have previous results that do suggest
this is the case.
JOSE LUIS CARRASCO JIMENEZ (jlcarrasco at ibmcp.upv.es) wrote:
: Dear colleagues,
: I am doing a three-hybrid screening in yeast. After
: retransformation of the rescued plasmids into yeast
: I am getting unexpected results, namely that the
: level of activation is lower and now it seems
: unspecific. This is quite surprising to me, since
: I have isolated twice the same cDNA, I mean, two
: of my positives beared the same cDNA insert, what
: I had interpreted as a good sign.
: Any help or comment will be highly appreciated.
: Many thanks in advance.
: Best wishes,
: Jose L. Carrasco
:jlcarrasco at ibmcp.upv.es