Hello: This is my 5-FOA recipe. Cheers, Greg Stuart :-)
1 mg/ml 5-Fluoroorotic Acid (5-FOA) Synthetic Dextrose Plates
Note. The pH of the 5-FOA medium is critically important (more acidic
is better)! Adjusting the pH to the standard yeast media of 5.8
renders the 5-FOA virtually useless resulting in very high
"background" growth), since only the protonated (low-pH) form is
permeable to yeast cells. Therefore, it is best to not adjust the pH
when making 5-FOA plates: the final pH will be pH ~3, but yeast
generally prefer growth in somewhat acidic conditions better anyway
=2E..
Materials:
1. Yeast synthetic drop-out medium supplement without uracil (DO
Supplement -Ura): Sigma Cat. no. Y-1501; add 1.92 g per liter of
medium. (I actually now use a home-made DO-Ura powder.)
2. Minimal SD Base: Clontech cat. no. 8602-1; contains yeast nitrogen
base (YNB), ammonium sulfate [A.S.; (NH4)2SO4)], and dextrose; use
26.7 g Min SD base + appropriate DO Supplement per liter of medium.
You can easily make your own Minimal SD Base by mixing 6.7 g of YNB
-AA +AS per 20 g D-glucose (dextrose).
3. Bacteriological agar: Sigma cat. no. A-5306; use 20 g per liter of
medium; note that extended autoclaving will result in soft agar.
4. 5-Fluoroorotic acid, monohydrate (5-FOA): Toronto Research
Chemicals cat. no. F595000. Other cheap sources (i.e., not Sigma!) are
US Biologicals, ZymoResearch, ...
SOLUTION A:
10.68 g Minimal SD base (Clontech; contains YNB, (NH4)2SO4, dextrose)
=20
0.77 g DO Supplement -URA
6.6 ml 20 mM (2.42 mg/ml) uracil (plate final: 0.040 mg/ml =3D 40 =B5g/ml)
=20
193.4 ml dH2O
=20
200 ml, total, in a 400 ml bottle equipped with a magnetic stir-bar
=20
1. Heat in microwave ~50-55 sec. ---> ~50-60=BAC.
2. Add 0.40 g 5-FOA powder. Stir; the 5-FOA will dissolve within
several minutes (generally almost immediately).
3. Do *not* adjust the pH (e.g., to 5.8) - see comments above! The pH
will initially be pH ~4.5 prior to addition of the 5-FOA, and pH ~2.9
after addition of the 5-FOA powder.
4. Place the solution in a 55=BAC water bath.
5. Be sure to filter-sterilize (0.20 =B5m; cellulose nitrate membrane)
SOLUTION A, prior to adding it to SOLUTION B!
Note: It generally recommended that YNB solutions be
filter-sterilized, rather than autoclaved.
SOLUTION B:
=20
8.0 g bacto-agar
=20
200 ml dH2O in a 400 ml bottle.
=20
1. Autoclave 20 min; mix well (to fully melt, suspend the molten agar;
will be very viscous) ---> 55=BAC bath.
2. Note: 'Over-autoclaving' can result in soft agar plates.
Plates:
1. Add warm (~55=BAC), filter-sterilized Solution A to Solution B. Mix
well, and pour plates.
2. Try to minimize bubbles during mixing (virtually impossible to
avoid; try to pour down the inside of the bottle, then swirl to mix
gently), that can be quite resistant to dispersion by flaming.
3. Should get ~12-14 10-cm diameter plates per 400 ml of molten 5-FOA
medium.
4. Protect the plates from light. Store at 4=BAC.
=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D=3D
On 22 Apr 2003 18:26:25 +0100, Martin Hirst <hirst at interomex.com>
wrote:
>Are you sure that your plates are correct - how are you making them? Try
>patching a ura+ and ura- strain onto the your plates to be sure they are o=
k.
>Generally we plate 10-100,000 cfu's on a large plate (20cm).
>>Cheers
>>Martin
>>> Have been trying to use 5FOA to dedelect for URA+ to no avail. I've
>> tried adding proline to the media in order to make the cells
>> hypersensitive to 5FOA and have also tried replica plating to new 5FOA
>> plates after initial growth.
>>=20
>> The problem is that after each try, instead of only a few cells
>> growing, I find a lawn of cells. How many cfu/ml should I be spreading
>> in the first place, and also is there anything I might be missing?
>>=20
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