Yeast Two Hybrid Project

[Giadoura]

Hi Everyone!
I'm new to this forum and I just have a question for you all.. if someone could be nice enough to answer me.. i am having some problems
I just started working on a yeast two hybrid project using the LexA system. My problem begins here: First I froze the co-transformed yeasts (i.e. with bait and library) in TE buffer/Glycerol with MgSO4 and then I titrated. At 1:10 to 1:10000 dilutions there are lots of yeast cells (microscope confirmed that smear had yeast). My problem is that at a 100,000 dilution (next 10-fold dilution), there is no yeast growth AT ALL.
Please someone tell me why there is lots of growth in the more concentrated one than at the diluted sample? I am a LOST student...
HELP please
THANKS

[kiki06]

Welcome to the forum!!
It may be that at high concentrations the yeast are actually dying off. The yeast are releasing enough amino acid (I'm assuming you're selecting using an auxotrophic marker) to allow growth of non transfected cells. Therefore it is only at these very high dilutions that you are really selecting for transformants. Yeast is very good at cannibalising materials for growth from other cells in the culture dying. This might be an explanation...

[dhruva]

does any body can give me notes onthis topic for my seminar? .
i m very much worried

please help me


my topic: 1.Molecular methods in diagnostic microbiology.
2.yeast two hybrid system.