13C source for protein NMR

Randall Willis willis at gandalf.psf.sickkids.on.ca
Mon Jul 29 08:26:20 EST 1996


Matthew,

Our lab routinely performs 15N/13C growths for NMR samples and we have 
worked with 13C-acetate in the past.  While the acetate is about half 
the price of glucose, we found that our final protein yields were also 
reduced by ~50%, so it was something of an even trade off.  Depending 
upon how well your protein expresses in minimal media, this may be okay 
for you as you may be able to get good samples from the lower yield or 
you may not see a difference with the expression in acetate.

Typically, our yields were reduced for two reasons; the bacteria don't 
express as well on acetate and the overall OD's tend to be lower.  I 
would recommend that you perform a number of growths with both carbon 
sources and take it from there.

When we perform a growth, I usually perform a fresh transformation of my 
plasmid into the BL21(DE3) and then grow a colony in LB for a few hours 
to overnight.  This is then used as an inoculum for the M9 growth which 
I induce anywhere from OD600=0.4 to 1.2 depending upon the construct and 
the culture.  If you go with the acetate, you may want to perform an 
intermediate growth of M9-glucose before going into M9-acetate as we 
have found that the bugs behave better if you give them only one 
challenge per step (ie rich media to minimal...good carbon to poorer).

If you need any more advice, drop a line.  Good luck.

Randall C Willis, Researcher        Publisher
(lab of Julie Forman-Kay)											Aliquotes Press
Biochemistry Research               "ALIQUOTES:A Journal of Molecular &
Hospital for Sick Children             Biochemical Humour"
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willis at gandalf.psf.sickkids.on.ca   rogerb at microsoft.com





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