Protocol Online logo
Top : New Forum Archives (2009-): : Molecular Biology

5-FOA media not letting ANY cells grow... help! - (Oct/11/2011 )

Hey people:

I'm a physics major turned biologist, so I know very little about biology. I'm currently doing a pop-in/pop-out protocol that involves integrating a plasmid with a truncated version of my gene of interest, then using 5-FOA media to "pop-out" the plasmid to get either the wild-type gene or the mutated gene (read Boecke "5-Fluoroorotic acid as a selective agent in yeast molecular genetics").

I pregrew my integrated colonies in YPD media and then plated them on 5-FOA along with a negative control and a positive control. The plates were at 30 degrees for several days, but nothing has grown. NOTHING. Not even on my positive control, which should have hundreds of colonies growing on it!

Any possible explanation for this? Could it be something wrong with the recipe I used? I used the recipe in Boecke's paper mentioned above. I'm not sure why there is nothing growing at all! It seems unusual.

Thanks in advance for your help!

-Kelsey Elaine Gray-

What is your positive and negative control? Are you sure that the concentration of 5-FOA is right? When you plate them on 5-FOA are there any other antibiotics in your medium?