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 < [email protected]> 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 > >
