no yeast growth - no yeast growth beyond a 1:10000 dilution (Jun/27/2005 )

Hi,

I am just embarking on a yeast-two-hybrid project using the LexA system. I am new to yeast work and am facing a silly problem, for which i can't seem to find a logical answer. I froze the co-transformed yeasts (i.e. with bait and library) in TE buffer/Glycerol with MgSO4 and did a titering. At 1:10 --> 1:10000 dilutions there are lots of yeast cells (seen as a smear and confirmed to be yeasts down the microscope). But at a 100,000 dilution (i.e. the next 10-fold dilution), there is absolutely no yeast growth. What could be the reason given that there is lots of growth in the more concentrated ones. I am at a loss! and would be thankful if someone can provide a logical explanation.
Thanks in advance.

G

This may be a possible explanation. At very high concentrations of yeast the dying cells themselves release enough amino acid (I am assuming you are selecting using an auxotrophic marker) to allow growth of non transfected cells. 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. I don't know if this really is the explanation but if the cell number is high enough.......???

What are you diluting into? Possibly some essential medium component is diluted
beyond the point of growth? A vitamin?

Terribly sorry...getting pple mixed up....OR I don't know how to use the website well enuff.

To Ajames: Your explanation seems Ok, just that the initial selection of cotransformants was done on triple selection plates (i..e. selecting for only the co-transformants I want)...so the transformants should have survived right?....so how come they don't appear after freezing and diluting?

To phage434: 1x TE was used to as a diluent, and 1xTE + Glycerol/MgSO4 was used to freeze down the cells.

Thanks for your help so far, but I am now re-doing my yeast-2-hybrid, this time selecting for Leu expression as well (an escape route from having to titer)...this time running into new problems. I should really start a new thread for these problems.

Regards,
G

If I were a yeast, I wouldn't like to be diluted in TE, but what do I know. You will be removing Ca++ and Mg++ ions from solution, which may be critical to their survival. But I don't work with yeast. Why are you diluting in TE?

Hi from my experience, titres can sometimes be like that. My advice, stop trying to find out why its like that, just repeat it and if it still shows the same, then I would just try to stop analysing it, somewhere the cells have just died

Thanks

Hi Propeller,

I started the message thread sometime back, but I did repeat the expt and managed to get some results.

Regards,
ggUss