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Certiprep 6850 freezer mill

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The CertiPrep 6850 Freezer/Mill is a laboratory equipment designed for cryogenic grinding and pulverization of samples. It features a temperature-controlled grinding chamber that can be cooled to cryogenic temperatures, allowing for efficient size reduction of materials that are otherwise difficult to grind at room temperature.

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Lab products found in correlation

Lp0037b, by Thermo Fisher Scientific (3 mentions) Yeast extract, by BD (1 mentions) Ultra edta free protease inhibitor, by Roche (1 mentions) Protease inhibitor cocktail, by Merck Group (1 mentions)

4 protocols using certiprep 6850 freezer mill

1

Yeast Cell Lysis and Protein Purification

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The Saccharomyces cerevisiae strains used in this study are shown in Table S5. Yeast cells were grown at 25°C in YP medium (1 % Yeast Extract, 21275, Becton Dickinson; 2 % bacteriological peptone, LP0037B, Oxoid) supplemented with 2 % Raffinose. In each case, a 12-litre exponential culture was grown to 2-3 x 107 cells/ml and then induced for 6 h at 20°C by addition of Galactose to a final concentration of 2 %. Cells were collected by centrifugation and washed once with lysis buffer (indicated below for each purification) lacking protease inhibitors. Cell pellets (~ 30 g) were then resuspended in 0.3 volumes of the indicated lysis buffer containing protease inhibitors. The resulting suspensions were then frozen dropwise in liquid nitrogen and stored at – 80°C. Subsequently, the entire sample of frozen yeast cells were ground in the presence of liquid nitrogen, using a SPEX CertiPrep 6850 Freezer/Mill with 3 cycles of 2’ at a rate of 15. The resulting powders were then stored at – 80 °C.
Xia Y., Sonneville R., Jenkyn-Bedford M., Ji L., Alabert C., Hong Y., Yeeles J.T, & Labib K.P. (2023). DNSN-1 recruits GINS for CMG helicase assembly during DNA replication initiation in C. elegans. Science (New York, N.Y.), 381(6664), eadi4932.
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2

Yeast Cell Lysis and Protein Purification

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The Saccharomyces cerevisiae strains used in this study are shown in Appendix Tables S1–S2. Yeast cells were grown at 25°C in YP medium (1% Yeast Extract, 21275, Becton Dickinson; 2% bacteriological peptone, LP0037B, Oxoid) supplemented with 2% Raffinose. In each case, a 12‐litre exponential culture was grown to 2‐3 x 107 cells / ml and then induced for 6 h at 20°C by addition of galactose to a final concentration of 2%. Cells were collected by centrifugation and washed once with lysis buffer (indicated below for each purification) lacking protease inhibitors. Cell pellets (˜ 30 g) were then resuspended in 0.3 volumes of the indicated lysis buffer containing protease inhibitors. The resulting suspensions were then frozen dropwise in liquid nitrogen and stored at – 80°C. Subsequently, the entire sample of frozen yeast cells were ground in the presence of liquid nitrogen, using a SPEX CertiPrep 6850 Freezer/Mill with 6 cycles of 2’ at a rate of 15. The resulting powders were then stored at −80°C. Details for each protein are given in Appendix Materials and Methods.
Xia Y., Fujisawa R., Deegan T.D., Sonneville R, & Labib K.P. (2021). TIMELESS‐TIPIN and UBXN‐3 promote replisome disassembly during DNA replication termination in Caenorhabditis elegans. The EMBO Journal, 40(17), e108053.
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3

Purification of Smc5/6 Complex

Cited 1 time
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Purification of the Smc5/65mer was as described previously (5 (link)). S. cerevisiae cells containing the overexpression constructs for five subunits of the Smc5/6 complex, wherein the Smc5 subunit is tagged with CBP tag, were grown at 30 °C in YP-GL (yeast extract, peptone, 2% glycerol, and 2% lactic acid) media until the log phase. Protein expression was induced by the addition of 2% galactose for 4 h. Harvested cells were resuspended in buffer E (45 mM Hepes-KOH pH 7.6, 10% glycerol, and 0.02% NP40) supplemented with 100 mM NaCl, 1 mM DTT, protease inhibitor cocktail (Sigma), and Ultra EDTA-free protease inhibitor (Roche) and then broken by using a freezer mill (SPEX CertiPrep 6850 Freezer/Mill). Cell powder was resuspended with buffer E supplemented with 300 mM NaCl and 1 mM DTT before being centrifuged for 30 min at 40,000 rpm to uncover the supernatant. Complexes containing Smc5-CBP fusion in the supernatant were pulled down by adding 2 mM CaCl2 and incubated with calmodulin resin for 2 h at 4 °C. After washing the resins with 10 bed volume of buffer E supplemented with 300 mM NaCl, 2 mM CaCl2, and 1 mM DTT, proteins were eluted using the same buffer without CaCl2 and supplemented with 1 mM EDTA and 2 mM EGTA. Peak fractions were pooled and subjected to gel-filtration on a Superose 6 Increase column. Peak fractions were collected and snap-frozen for storage.
Li S., Yu Y., Zheng J., Miller-Browne V., Ser Z., Kuang H., Patel D.J, & Zhao X. (2023). Molecular basis for Nse5-6 mediated regulation of Smc5/6 functions. Proceedings of the National Academy of Sciences of the United States of America, 120(45), e2310924120.
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4

Yeast Cell Lysis and Protein Purification

Check if the same lab product or an alternative is used in the 5 most similar protocols
The Saccharomyces cerevisiae strains used in this study are shown in Table S5. Yeast cells were grown at 25°C in YP medium (1 % Yeast Extract, 21275, Becton Dickinson; 2 % bacteriological peptone, LP0037B, Oxoid) supplemented with 2 % Raffinose. In each case, a 12-litre exponential culture was grown to 2-3 x 107 cells/ml and then induced for 6 h at 20°C by addition of Galactose to a final concentration of 2 %. Cells were collected by centrifugation and washed once with lysis buffer (indicated below for each purification) lacking protease inhibitors. Cell pellets (~ 30 g) were then resuspended in 0.3 volumes of the indicated lysis buffer containing protease inhibitors. The resulting suspensions were then frozen dropwise in liquid nitrogen and stored at – 80°C. Subsequently, the entire sample of frozen yeast cells were ground in the presence of liquid nitrogen, using a SPEX CertiPrep 6850 Freezer/Mill with 3 cycles of 2’ at a rate of 15. The resulting powders were then stored at – 80 °C.
Xia Y., Sonneville R., Jenkyn-Bedford M., Ji L., Alabert C., Hong Y., Yeeles J.T, & Labib K.P. (2023). DNSN-1 recruits GINS for CMG helicase assembly during DNA replication initiation in C. elegans. Science (New York, N.Y.), 381(6664), eadi4932.
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