Thiamine in M9 minimal medium

Emir Khatipov ekhatipo at NOSPAMmidway.uchicago.edu
Mon Sep 18 22:33:19 EST 2000


Hi,
I have problems with growing TOP10 transformants in M9 minimal medium.

Could someone experienced in using M9 minimal medium advise me what is the
most appropriate way of preparing and handling/storing thiamine solution.
What should be the concentration of the stock solution so that thiamine will
dissolve readily in water, and whether it should be prepared fresh or can be
stored, how long? I was advised to add up to 10ug/ml thiamine to the medium,
and I make 1000 x water solution by mixing thiamine crystals with water and
adding alkali.

The TOP10 cells I am using cells that are L-lysine auxotrophs, so any
additional comments on lysine solution would be appreciated, too (making,
storing).

I make the M9 medium according to conventional protocols. Since there is
nothing special about M9 salts and glucose, I don't think I am screwing
something up there. I guess my cells do not grow because I do something
wrong when making thiamine or lysine.

I know thiamine is light-sensitive and I store it in the dark. Anyway, I
could not get my cells grow even with freshly made thiamine solution.

I also know that I could add up to ~4% LB to facilitate growth, but I do not
want to do that.

Thanks.
Emir








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