P.S. you can easily enough kill E. coli with excess selenium! Failure to grow is often observed wgen SeMet is used in excess of 160mg/L (total, so if you're adding a D/L mix it is still 160mg/ml een though you're only adding 80mg D and 80mg L).
A. - Cosmic Cats approve of this message On Sun, Feb 15, 2015 at 10:20 AM, Artem Evdokimov <artem.evdoki...@gmail.com > wrote: > The "Doublie protocol" usually refers to metabolic suppression of > methionine biosynthesis by means of adding excess amounts of certain amino > acids to a medium that is innately methionine-free (doesn't have to be > minimal, in fact rich medium is better), then replacing Met with SeMet. > Yeast extract has appreciable quantities of methionine, however you did not > mention whether you have added the extra four amino acids as well? Without > them, non-auxotrophic E.coli will make their own methionine. > > The reason your culture did not grow (assuming that your M9 medium is > correctly assembled and correctly pH-balanced) could be any number of > things - did you for example autoclave it with glucose in the medium? Did > you use a very small inoculum (sometimes it is better to use large inoculum > volume, that is spun down to avoid the transfer of original rich medium), > etc. etc. > > Best regards, > > Artem > > - Cosmic Cats approve of this message > > On Sat, Feb 14, 2015 at 4:26 PM, Giulliana Rangel < > giulliana.ran...@gmail.com> wrote: > >> Dear all, >> >> >> I am trying to express my protein in minimal medium M9 (trace elements + >> biotin and thiamine, according to Doublie protocol) but, after O.N. >> incubation there wasn't growing.I tried to change vitamins concentration >> and in the end just had good results when I put 0,1% yeast extract. >> >> So, for this I would like some help... there is in yeast extract large >> quantities of methionine? Because, if yes it can put down my essay with >> semet. >> >> P.S >> >> 1. I put the bacterials first in L.B, then in solid minimal medium >> M9 where they growed normally and when i inoculate in the same minimal >> medium M9 composition but liquid form, there wasn't growing. >> 2. My e.coli strain is roseta (de3) >> >> >> Thanks in advance >> >> Best Regards, >> >> Giulliana >> >> >> >> -- >> Giulliana Rangel >> M.S. Biotechnology Program - UNIFESP >> Structural Biology Laboratory >> Phone: +55 (12) 3309-9698 >> 330 Talim St >> ZIP CODE 12231-280 >> São José dos Campos - SP/ BR >> >> >
