Buffered Glycerol Complex Medium, Sterile 2.0% Peptone, 1.0% Yeast extract, 1.34% Potassiumphosphate pH 6.0, 1.34% Yeast nitrogen base w/o AA, 0.4 mg/L Biotin, 1.0%

Name BMGY-Buffered Glycerol-Complex Medium Kit Product number B8000 1.2 Relevant identified uses of the substance or mixture and uses advised against Relevant identified uses General use 1.3 Details of the supplier of the safety data sheet Teknova 2290 Bert Dr. Hollister California 95023 United States Telephone: 831-637-1100 Telefax: 831-637-2355 buffered glycerol-complex medium (BMGY; 10 g/L of yeast extract, 20 g/L of peptone, 13.4 g/L of YNB, 4 £ 10¡4 g/L of biotin, 10 mL/L of glycerol and 0.1 mol/L of potassium phosphate buffer, pH 6.0) shaken at 250 rpm for 18 h at 30 C. The cell pellets were harvested and resuspended in 50 mL of buffered methanol-complex Mut s colonies (10) were then used to inoculate 10 ml buffered minimal glycerol-complex medium (BMGY) pH 6 (0. 1 M potassium phosphate buffer pH 6. 0, 1% yeast extract, 2% peptone, 1. 34% YNB without amino acids, 0. 4 μg ml −1 d-biotin, 1% glycerol) in a 50-ml loosely capped tube, shaken at 250 rpm for 2 days at 30°C, the cells were Jun 01, 2020 · YPD broth (Yeast extract peptone dextrose broth, 10 g/L yeast extract, 20 g/L peptone and 20 g/L dextrose) and BMGY (Buffered glycerol-complex medium, 10 g/L yeast extract, 20 g/L peptone, 40 ml/L glycerol, 1.34 % (w/v) YNB, 4 × 10 −5 % (w/v) biotin and 100 mM potassium phosphate buffer pH 6.0) medium were used for pre-culture and the Precultures were prepared in Buffered Glycerol Complex Medium (BMGY) in 500‐mL shake flasks, with the following composition (per L): 10 g glycerol, 10 g yeast extract, 20 g peptone, 1.34 % (v/v) yeast nitrogen base (YNB), and 0.004 g biotin in 100 mM potassium phosphate buffer, pH 6.0. Preparation of inoculum I wish to know the role of biotin in BMGY (Buffered Glycerol Complex Medium) Other articles mostly add it depending on the dissolved oxygen or methanol in the medium, but we do not have way to

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All other chemicals used were analytical grade reagents unless otherwise stated. Yeast extract peptone dextrose (YPD) medium, buffered glycerol complex (BMGY) medium, and buffered methanol complex (BMMY) medium were prepared according to the manual of Pichia Expression Kit (Version F, Invitrogen). P. pastoris GS115 cells transformed with pHIL‐D2‐ACMSD I were incubated in buffered glycerol complex medium, at 30 °C, up to an A 600 of 3.0. The culture was centrifuged at 5000 g for 10 min (Sorvall centrifuge RC5B plus, Superlite GSA rotor) and the cell pellet was resuspended in one‐fifth of the original culture volume of buffered

All other chemicals used were analytical grade reagents unless otherwise stated. Yeast extract peptone dextrose (YPD) medium, buffered glycerol complex (BMGY) medium, and buffered methanol complex (BMMY) medium were prepared according to the manual of Pichia Expression Kit (Version F, Invitrogen).

Twenty transformants were used to inoculate 40 ml of buffered glycerol complex medium (BMGY) (Invitrogen) (0.1 M potassium phosphate buffer at pH 6.0 containing 1% [wt/vol] yeast extract, 2% [wt/vol] peptone, 1.34% [wt/vol] yeast nitrogen base without amino acids, 1% [vol/vol] glycerol, and 4 × 10 −5 % [wt/vol] biotin). Cultures were grown Then, the culture was transferred into 1 L fresh BMGY, and all of the cells were collected by centrifugation at room temperature (3,500 g for 5 min) until the of the culture reached approximately 6, at which point it was resuspended in 100 mL of buffered methanol-complex medium (BMMY). Methanol (3%) was added to the media daily to induce the Jul 27, 2017 · The cells were then recovered by adding 500 µL liquid LB medium and incubating at 37 °C for 1 h and then plated on LB plates supplemented with 25 μg/mL Zeocin (TFS, R25001). After incubated at 37 °C overnight, ten colonies were cultured in 3 mL liquid LB medium supplemented with 25 µg/mL Zeocin at 37 °C overnight. The recombinants were cultured in Buffered Glycerol- complex Medium (BMGY) at 30℃ and then resuspended in Buffered Methanol-complex Medium (BMMY). The cultures were shaken at 250 r/min for 108 h at 29℃, and the methanol concentration retained 0.5% (w/v). Column chromatography The supernatant was harvested by 3600 rpm centrifugation grown in 1 L of buffered glycerol complex medium to an optical density over 6 at 600 nm. Cells were induced with 1% methanol by replacing the buffered glycerol complex medium with buffered methanol complex medium. Methanol was added at a concentra-tion of 1% every 24 hours during the induction phase (up to 72 hours). Cultures were centrifuged All other chemicals used were analytical grade reagents unless otherwise stated. Yeast extract peptone dextrose (YPD) medium, buffered glycerol complex (BMGY) medium, and buffered methanol complex (BMMY) medium were prepared according to the manual of Pichia Expression Kit (Version F, Invitrogen). P. pastoris GS115 cells transformed with pHIL‐D2‐ACMSD I were incubated in buffered glycerol complex medium, at 30 °C, up to an A 600 of 3.0. The culture was centrifuged at 5000 g for 10 min (Sorvall centrifuge RC5B plus, Superlite GSA rotor) and the cell pellet was resuspended in one‐fifth of the original culture volume of buffered