Autoclaving amino acids and sugars

Robert Nakamura

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Feb 11, 1996, 3:00:00 AM2/11/96

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Hi,

I am trying to cut down on contamination in our lab by autoclaving as many
components of media as possible. Will autoclaving galactose and sucrose
form unacceptable levels of glucose? Also, we have always autoclaved
threonine and aspartic acid; Fred Sherman writes in the Guide to Yeast
Genetics that these should be added separately. Any idea why? Thanks in
advance,

Robert Nakamura
Northwestern University

Matthew Sachs

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Feb 12, 1996, 3:00:00 AM2/12/96

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I can share what we use for routine testing of the requirements of
vegetatively growing Neurospora crassa for fourteen amino acids in
supplemented minimal medium. David Perkins provided this information in a
course he taught at Stanford University. Amino acid stocks are prepared in
water and kept refrigerated with a drop of chloroform to preserve them.
The mg/ml of amino acid in the stock, and the concentration factor of the
stock are indicated.

L-arginine (40 mg/ml; 80X)
L-asparagine (20 mg/ml; 50X)
L-aspartic acid (50 mg/ml; 50X)
L-glutamine (20 mg/ml; 50X)
L-histidine-HCl (25 mg/ml; 50X)
DL-isoleucine (3 mg/ml; 33X)
L-leucine (5 mg/ml; 25X)
L-lysine (20 mg/ml; 40X)
L-methionine (10 mg/ml; 20X)
L-phenylalanine (10 mg/ml; 50X)
L-proline (10 mg/ml; 20X)
L-serine (50 mg/ml; 125X; protect from light)
L-threonine (5 mg/ml; 50X)
L-tryptophan (8 mg/ml; 40X)

-----------------------------------------------------------
Matthew Sachs
Department of Chemistry, Biochemistry and Molecular Biology
Oregon Graduate Institute of Science and Technology
20000 NW Walker Road
P.O. Box 91000
Portland, OR 97291-1000
503 690-1487 Phone
503 690-1464 Fax
msa...@admin.ogi.edu

Kevin Morano

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Feb 13, 1996, 3:00:00 AM2/13/96

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Hi,

You can autoclave galactose, but don't autoclave sucrose unless some
glucose is OK. We routinely filter sterilize all our amino acids.
Overkill, but at least we know they will work.

Kevin

Rich Buckholz

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Feb 14, 1996, 3:00:00 AM2/14/96

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In article <robnak-1102...@triploid.ls.nwu.edu>,
rob...@casbah.acns.nwu.edu (Robert Nakamura) wrote:

> Hi,
>
> I am trying to cut down on contamination in our lab by autoclaving as many
> components of media as possible. Will autoclaving galactose and sucrose
> form unacceptable levels of glucose? Also, we have always autoclaved
> threonine and aspartic acid; Fred Sherman writes in the Guide to Yeast
> Genetics that these should be added separately. Any idea why? Thanks in
> advance,
>
> Robert Nakamura
> Northwestern University

In my experience, autoclaving galactose and sucrose stocks produces an
unacceptably high level of repressing glucose. Try filter sterilizing. I
don't have any first hand experience with thr and asp, except that we make
our dropout media from a mixture of ground up amino acids (including asp
and thr) which is added to media and then autoclaved at 121C for 15 min.
This has worked fine for years, although our bugs aren't asp or thr
auxotrophs so we don't know for sure it's doing anything.

Rich Buckholz

Rafael Maldonado

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Feb 16, 1996, 3:00:00 AM2/16/96

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Some aminoacids can be autoclaved, other cannot.

Autoclave the following:
-arginine
-glycine
-histidine
-isoleucine
-leucine
-lysine
-methionine
-phenylalanine
-proline
-serine
-threonine
-valine

-adenosina
-guanosine
-uracil

Filter the following:
-asparagine
-aspartic acid
-cysteine
-glutamic acid
-glutamine
-tryptophan
-tyrosine

Yes, I know that many people autoclave tryptophan, for example. But the
health of those aminoacids is not good after autoclaving.

From: Advanced Bacterial Genetics (Davis, Roth and Botstein). 1980. CSHL
Press.

Rafa