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M9 minimal media

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M9 minimal media is a defined, inexpensive growth medium used for culturing bacteria, such as Escherichia coli, in laboratory settings. It provides the essential nutrients and salts required for bacterial growth and maintenance.

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Lab products found in correlation

13c labeled glucose, by Cambridge Isotopes (2 mentions) Bl21 ai cells, by Thermo Fisher Scientific (2 mentions) Chitin column, by New England Biolabs (2 mentions) 15n labeled ammonium chloride, by Cambridge Isotopes (2 mentions) Topspin, by Bruker (1 mentions) Nmrpipe, by Bruker (1 mentions)

4 protocols using m9 minimal media

1

Producing Deuterated Kai Proteins

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KaiA, KaiB, and KaiC originating from S. elongatus PCC 7942 were expressed in Escherichia coli as Strep-tagged recombinant proteins and purified as previously described21 (link). The expression plasmids of the KaiC mutants (KaiCDE, KaiCDT, KaiCAA, and KaiCSE) were also constructed according to a previous study21 (link). KaiCDE is a mutant of KaiC with aspartate and glutamate residues at positions 431 and 432, respectively, that mimics hyperphosphorylated KaiC, whereas KaiCAA is a mutant with alanine residues at these positions that mimics hypophosphorylated KaiC. For preparation of the deuterated proteins, the bacterial cells were grown in M9 minimal media containing glucose as a mixture with varying ratios of isotopically natural and fully deuterated glucose (1,2,3,4,5,6,6-D7, 98%, Cambridge Isotope Laboratories, Inc.), along with varying ratios of H2O and D2O as previously described23 (link)24 (link).
Sugiyama M., Yagi H., Ishii K., Porcar L., Martel A., Oyama K., Noda M., Yunoki Y., Murakami R., Inoue R., Sato N., Oba Y., Terauchi K., Uchiyama S, & Kato K. (2016). Structural characterization of the circadian clock protein complex composed of KaiB and KaiC by inverse contrast-matching small-angle neutron scattering. Scientific Reports, 6, 35567.
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2

Affinity Purification of NpR6012g4 Protein

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The protein sample in this study consists of 180 native residues (M583-G762, Fig. 1) after removal of a C-terminal intein-CBD tag used for affinity purification (Kim et al, 2012 (link)). NpR6012g4 was expressed in BL21-AI cells (Invitrogen) grown in M9 minimal media supplemented with ALA (100 μM), 15N-labeled ammonium chloride, and/or 13C-labeled glucose (Cambridge Isotopes) using a published system for induction of protein expression and chromophore biosynthesis (Gambetta & Lagarias, 2001 (link)). Affinity purification of NpR6012g4 using a chitin column (NEB) followed our previous procedure (Kim et al, 2012 (link); Rockwell et al, 2012 (link); Rockwell et al, 2015a (link); Rockwell et al, 2015b (link)). Peak eluted fractions were pooled for overnight dialysis into 10 mM sodium phosphate (pH 7.4) supplemented with 1 mM EDTA to remove residual metal ions followed by final overnight dialysis into 10 mM sodium phosphate (pH 7.4). The protein was concentrated to approximately 0.7 mM, and D2O was added to 7% (v/v). Dark reversion of the metastable green-absorbing state under these conditions was < 10% after 24 hours at 298 K as reported previously (Rockwell et al, 2015b (link)). All subsequent manipulations were performed on samples kept in darkness.
Lim S., Yu Q., Rockwell N.C., Martin S.S., Clark Lagarias J, & Ames J.B. (2015). 1H, 13C, and 15N Chemical Shift Assignments of Cyanobacteriochrome NpR6012g4 in the Green-Absorbing Photoproduct State. Biomolecular NMR assignments, 10(1), 157-161.
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3

Preparation and NMR Analysis of Labeled Proteins

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The 15 N-labeled S. cerevisiae ubiquitin, Polg 515, PCNA, and GB1 tag were prepared using a bacterial expression system and purification as previously described except in M9 minimal media containing 15 NH 4 Cl as the nitrogen source (Cambridge Isotope Laboratories, Andover, MA, USA). All samples for NMR experiments were prepared in buffer C containing 10% D 2 O. The two-dimensional 1 H- 15 N HSQC NMR spectra were acquired with a Bruker 700 MHz spectrometer (KBSI-Ochang) at 25 °C. The weighted chemical shift perturbations (Dd) for backbone 1 H and 15 N resonances were calculated by the following equation:
where DH and DN are, respectively, the chemical shift changes in the 1 H and 15 N dimensions [27] .
NMR data were processed with NMRPIPE [28] and TOPSPIN (BrukerBioSpin, Bruker, Billerica, MA, USA), and the spectra were analyzed with SPARKY (SPARKY 3, San Francisco, CA, USA) and NMRFAM SPARKY [29] . HSQC titrations were performed by mixing unlabeled into labeled protein at the indicated molar ratios. The concentration of protein at the start of titration was 0.8 mM. Ubiquitin backbone resonance assignments were obtained from BMRB ID 4769 and transferred to HSQC spectra by NMRFAM SPARKY.
Duong P.T.M., Bui A.T.N., Kim S.O., Park H.S., Seo Y.S, & Choi B.S. (2020). The interaction between ubiquitin and yeast polymerase η C terminus does not require the UBZ domain. FEBS letters, 594(11).
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4

Preparation of Labeled Photoreceptor Protein

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The protein sample in this study consists of 180 native residues (M583-G762, Fig. 1) after removal of a C-terminal intein-CBD tag used for affinity purification (Kim et al, 2012 (link)). NpR6012g4 was expressed in BL21-AI cells (Invitrogen) grown in M9 minimal media supplemented with ALA (100 μM), 15N-labeled ammonium chloride, and/or 13C-labeled glucose (Cambridge Isotopes) using a published system for induction of protein expression and chromophore biosynthesis (Gambetta & Lagarias, 2001 (link)). Affinity purification of NpR6012g4 using a chitin column (NEB) followed our previous procedure (Kim et al, 2012 (link); Rockwell et al, 2012 (link); Rockwell et al, 2015a (link); Rockwell et al, 2015b (link)). Peak eluted fractions were pooled for overnight dialysis into 10 mM sodium phosphate (pH 7.4) supplemented with 1 mM EDTA to remove residual metal ions followed by final overnight dialysis into 10 mM sodium phosphate (pH 7.4). The protein was concentrated to approximately 0.7 mM, and D2O was added to 7% (v/v). Dark reversion of the metastable green-absorbing state under these conditions is < 10% after 24 hours at 298 K as reported previously (Rockwell et al, 2015b (link)). All subsequent manipulations were performed in darkness.
Yu Q., Lim S., Rockwell N.C., Martin S.S., Lagarias J.C, & Ames J.B. (2015). 1H, 15N, and 13C chemical shift assignments of cyanobacteriochrome NpR6012g4 in the red-absorbing dark state. Biomolecular NMR assignments, 10(1), 139-142.
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