: Thursday, April 19, 2007 10:31 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Protein expression in Minimal media (M9)
Manish,
Assuming that you need the minimal medium for Se, have you tried the
regular heavy atom derivatives? You already have diffracting crystals...
M9 medium can be
Manish,
Assuming that you need the minimal medium for Se, have you tried the
regular heavy atom derivatives? You already have diffracting crystals...
M9 medium can be slightly difficult. However, no one says that you *have*
to use minimal medium such as M9 (again, assuming that you're doing
Se-Me
Are you adding vitamins to your M9 minimal? RosettaBlue is thiamin
requiring and can not grow in the absence of thiamin. The thiamin
requirement is so low that you can often get slow growth to a low OD
based on residual thiamin in the cells, but you will not get robust
growth.
Also, min
What we usually do is to grow cells in this media:
400ml H2O milli-Q Autoclaved
120ml AAmix (5X) Filtered
60ml M9 (10X) Autocl.
6ml TES (100X) Filtr.
12ml Glucose 20% Filtr.
0.6ml MgSO4 (1M) Autocl.
0.18ml CaCl2 (1M) Autocl.
1.2ml Biotine (5mg/ml) Filtr.
0.6ml Thiamine (10mg/ml)
Manish,
I also had a similar problem getting cells (C41 (DE3) in my case) to grow
in minimal media. To get around this problem, I took cells from an agar
plate and grew them in a small volume (5 mL) of the minimal media. Once
that culture got thick, I then inoculated 200 mL of minimal media with 0
I had a similar situation, i.e. the cells wouldn't grow past an OD of 0.1 on
minimal media, but in my case I did get protein expression. The way I got
around it was as follows. I grew the cells up in minimal media plus the
amino acids that suppress methionine biosynthesis, plus L-methionine. For
Hi,
from my experience with minimal media expression you're probably
better off starting with a good, thick rich media overnight growth,
and then diluting that out 1:20 – 1:50 into the M9.
Richard
--
Richard P. Grant
School of Molecular & Microbial Biosciences University of Sydney
