Ilv minus

Alex Brands abbrands at artsci.wustl.edu
Fri Jul 20 16:00:47 EST 2001


On 19 Jul 2001, Heriberto Correia wrote:
>             Hi
>             I have one yeast strain
>             (ade 2-40/ade 2-119, trp 5-12/trp 5-27, ilv1-92/ilv1-92)
>             And I need to prepare mininal mediun without ilv.
>             The yeast extract w/o aminoacids (Difco 0919-15)
>             Lacks of trp, met and his that means it contain the others
>             aminoacids.
>             How I can prepare mininal mediun without ilv?
>             Does anybody have an recipe?
>             ThanK you in advandce

I think you mean Difco Yeast Nitrogen Base without Amino Acids which has
the same catalog number (0919-15).  The recipe for synthetic media that I
use contains this product, and calls for the addition of isoleucine and
valine (which the ilv mutant requires).  I don't know why they explicitely
list the absence of his, met, and trp.  I have used this media for ura and
lys selection as well, so it obviously does not contain those chemicals
either.

The bottle says it is of known chemical composition.  If you can get your
hands on a Difco Manual, you can confirm wether or not it contains
isoleucine and valine.

Looking at our very old Difco manual (printed 1964) the only amino acids
listed in the ingredients for Yeast Nitrogen Base are His, Met, and Trp.
These are the same three that are explicitely listed as absent on the
label.  Perhaps that is why they make a point af saying that they are no
longer in there.  Since these are now commonly used as markers for yeast
transformation, that's probably why they are no longer included in the
mix.  I'm pretty sure that the product with catalog numner 0919-15 does
not contain any amino acids.

Here is the recipe I use:

100g yeast nitrogen base without amino acids
293g ammonium sulfate
1.2g adenine sulfate
1.2g L-arginine
1.8g L-isoleucine
1.2g L-methionine
3.0g L-phenylalanine
1.8g L-tyrosine
9.0g L-valine

mix very well

For 1 liter:

7.1g of the above mix
20g glucose
15g agar (if for plates)
1 NaOH pellet (should be pH 5.8 at the end)
1 liter H2O

To supplement (per liter)
20mg his
60mg leu
20mg trp
20mg ura
30mg lys

some people prefer to make the media in 800 mls, then autoclave, then add
200 mls 10% glucose (autoclaved seperately).  Autoclaving the glucose with
the other components results in a brownish color.  The longer you
autoclave, the darker it gets.

---





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