I'm a first year grad student, and here is my latest quandry.
I am trying to knockout an integrated URA marker in my yeast line in order that I may use URA to select for the addition of another plasmid which I have prepared.
I have succesfully done this with one of my yeast lines, by plating onto a 5'FOA plate.
Any colonies that grew on the first 5'FOA plate were patched onto a second 5
'FOA plate simultaneously with a -URA plate.
Colonies that did grow on the second 5'FOA plate but not on the -URA plate were considered to NOT contain the URA marker anymore.
With the second yeast line that I am working on it has not been so easy. The yeast will commonly grow on the second generation 5'FOA plate AND the -URA plate, indicating the presence of some resistance to 5'FOA that is independent of the presence of URA marker.
In my most recent attempt, I noticed that the colonies on the second generation 5'FOA plate are all white, whereas the colonies on the URA plate are pink. Is there any signfiicance to this?
Any general tips about how YOU use 5'FOA for selection?
On advice of my mentor i have been transforming in a linear piece from the pRS316-URA containing plasmid to force recombination before plating on the first 5'FOA plate.