Problem with 5-FOA plates.

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Mon Sep 25 07:39:32 EST 1995


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In a message dated 95-09-19 14:09:23 EDT, BIOSCI-REQUEST at net.bio.net writes:

>Subj:	RE: Problem with 5-FOA plates.
>Date:	95-09-19 14:09:23 EDT
>From:	nmorey at bimcore.emory.edu (Natalie Jean Morey)
>Resent-from:	BIOSCI-REQUEST at net.bio.net
>To:	yeast at net.bio.net
>
>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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>Date: 19 Sep 1995 13:54:29 GMT
>From: nmorey at bimcore.emory.edu (Natalie Jean Morey)
>Subject: RE: Problem with 5-FOA plates.
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