Problem with 5-FOA plates.

Natalie Jean Morey nmorey at bimcore.emory.edu
Tue Sep 19 08:54:29 EST 1995


Kenneth Shaw (ez051834 at rocky.ucdavis.edu) wrote:
: My 5-fluorooratic acid plates are not eliminating yeast carrying a plasmid 
: on which the ura-3 gene resides. Maybe someone could review my prepartion 
: protocol and tell me what I am doing wrong. Does 5-FOA go bad when stored as a
: powder at -20 C.

: 1 g 5-FOA
: 100 ml H2O
: 100 ml 10xUra
: 100 ml 10xTrp
: 100 ml 10xAA -Ura, -Trp, -His
: 100 ml 20% galactose
: 0.5 ml 40% glucose
: -stir with low heat (~1 hr) until 5-FOA dissolved
: -filter sterilize
:  
: 6.7 g YNB -AA/AS
: 5 g ammonium sulfate
: 20 g bacto agar
: 1 pellet NaOH
: 475 g H20
: -Autoclave 1 hr
: -cool to 50 C and add 5-FOA mix
: -swirl to mix and pour

: My problem is that seven of eight yeast colonies that have been grown on 
: this medium will then still grow on minimal medium plates without uracil. 
: Please post suggestions here or to my email address: kjshaw at ucdavis.edu. Thank
: you for your time.


	Have you tried purifying the paps on YPD first?  I had problems 
that I had some mixed paps, so when you try to grow selectively, the few 
that are still carrying a plasmid grow and make it look like the whole 
patch still had it.  Also, if you are picking patches to 5-FOA, make sure 
you don't pick too many cells.
	As for the media and storing 5-FOA, it looks very similar to ours 
(except for the galactose and the NaOH pellt), and we store our 5-FOA at 
4 degrees.  
	Hope this helps.
	
	Natalie Morey
	nmorey at bimcore.emory.edu
	Emory University
	Atlanta, GA




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