The culture media and meiotic time course were essentially performed as previously described (Kim et al., 2010 (link)). Cells were patched to YPG plates (1% yeast extract, 2% peptone, 3% glycerol, and 2% bactoagar) for 24 h at 30°C. To select single colonies, cells from the YPG plate were streaked onto YPD plates (1% yeast extract, 2% peptone, 2% glucose, and 2% bactoagar) and grown at 30°C for 2 days. A single diploid colony resulting from this streaking was inoculated into 2 ml liquid YPD medium (1% yeast extract, 2% peptone, 2% glucose) and incubated at 30°C for 24 h. For synchronous meiosis, YPD cultures were inoculated in SPS medium (1% potassium acetate, 1% bactopeptone, 0.5% yeast extract, 0.17% yeast nitrogen without amino acids, 0.5% ammonium sulfate, 0.05 M potassium biphthalate, and 2 drops/L antifoam [Sigma, USA], pH 5.5) at a 1:500 dilution and cultured for 18 h. Meiosis was initiated in SPM medium (0.2% potassium acetate, 0.02% raffi-nose, and 2 drops/L antifoam). Meiotic cells were harvested and resuspended in 50 mM Tris-HCl and 50 mM EDTA. Cross-linking of cells was performed with psoralen under UV light for 10 min.
Antifoam
Manufactured by Merck Group
Sourced in United States, Germany
Antifoam is a laboratory equipment product designed to prevent or reduce the formation of foam in various liquid-based processes. It functions by breaking down and suppressing the surface tension of foams, allowing for better control and management of liquid handling during experiments or industrial operations.
Automatically generated - may contain errors
Lab products found in correlation
Carboxymethylcellulose, by Merck Group (1 mentions)
D luciferin, by PerkinElmer (1 mentions)
Polyvinylpyrrolidone, by Merck Group (1 mentions)
Sodium dodecyl sulfate (sds), by Serva Electrophoresis (1 mentions)
Bradford solution, by Bio-Rad (1 mentions)
Pwo dna polymerase, by Roche (1 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by Avantor (1 mentions)
F1 6bp, by Merck Group (1 mentions)
Benzonase, by Merck Group (1 mentions)
Dl dithiothreitol, by Merck Group (1 mentions)
Dulbecco phosphate buffered saline (dpbs), by Corning (1 mentions)
Sarkosyl, by Merck Group (1 mentions)
Sera mag magnetic speedbeads, by GE Healthcare (1 mentions)
Biostat a, by Sartorius (1 mentions)
Inpro 6800, by Mettler Toledo (1 mentions)
Psoralen, by Merck Group (1 mentions)
12 protocols using antifoam
1
Synchronous Meiosis Induction in Yeast
2
Strain LF660 Cultivation in Bioreactor
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Strain LF660 was cultivated in the 10 L stirred tank reactor system (Biostat, Braun, Melsungen, Germany) with straight propellers using the same type of pre-cultures as for Erlenmeyer flask cultivation. Oxygen concentration, pH, and stirring speed were monitored. The oxygen content in the medium was controlled by adjusting stirrer speed and the aeration rate was set to a minimum of 30% air saturation. Foam formation was stopped by addition of antifoam (Sigma, Taufkirchen, Germany). After cultivation, cells were separated from the culture broth by means of centrifugation. For the 1 L and 10 L scale, culture supernatant and cells were extracted by the addition of 2 volumes ethyl acetate. The organic solvent was separated and concentrated to dryness under reduced pressure.
3
Compound Procurement for Research
4
Yeast Cultivation Protocols for Research
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Saccharomyces cerevisiae strains with uracil auxotrophy were grown on YPD plates containing 20 g/L glucose, 10 g/L yeast extract, 20 g/L peptone from casein and 20 g/L agar. Plasmid carrying strains were grown on selective growth medium containing 6.9 g/L yeast nitrogen base w/o amino acids (Formedium, Hunstanton, UK), 0.77 g/L complete supplement mixture w/o uracil (Formedium), 20 g/L glucose and 20 g/L agar. Shake flask cultivations were performed in minimal medium containing 30 g/L glucose, 7.5 g/L (NH4)2SO4, 14.4 g/L KH2PO4, 0.5 g/L MgSO4·7H2O, 2 mL/L trace element solution and 50 μL/L antifoam (Sigma-Aldrich, St. Louis, MO, USA). After sterilization, vitamin solution was added at a concentration of 1 mL/L. The batch phase medium during aerated bioreactor cultivations contained 10 g/L glucose, 5 g/L (NH4)2SO4, 3 g/L KH2PO4, 0.5 g/L MgSO4·7H2O, 1 mL/L trace element solution, 50 μL/L antifoam and 1 mL/L vitamin solution. During the fed-batch phase, the feed medium was five times concentrated and contained glucose to a concentration of 100 g/L. The composition of the trace element and vitamin solution has been reported by Verduyn et al. [33 (link)].
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Saccharomyces cerevisiae strains with uracil auxotrophy were grown on YPD plates containing 20 g/L glucose, 10 g/L yeast extract, 20 g/L peptone from casein and 20 g/L agar. Plasmid carrying strains were grown on selective growth medium containing 6.9 g/L yeast nitrogen base w/o amino acids (Formedium, Hunstanton, UK), 0.77 g/L complete supplement mixture w/o uracil (Formedium), 20 g/L glucose and 20 g/L agar. Shake flask cultivations were performed in minimal medium containing 30 g/L glucose, 7.5 g/L (NH4)2SO4, 14.4 g/L KH2PO4, 0.5 g/L MgSO4·7H2O, 2 mL/L trace element solution and 50 μL/L antifoam (Sigma-Aldrich, St. Louis, MO, USA). After sterilization, vitamin solution was added at a concentration of 1 mL/L. The batch phase medium during aerated bioreactor cultivations contained 10 g/L glucose, 5 g/L (NH4)2SO4, 3 g/L KH2PO4, 0.5 g/L MgSO4·7H2O, 1 mL/L trace element solution, 50 μL/L antifoam and 1 mL/L vitamin solution. During the fed-batch phase, the feed medium was five times concentrated and contained glucose to a concentration of 100 g/L. The composition of the trace element and vitamin solution has been reported by Verduyn et al. [33 (link)].
5
Optimized Chemicals Sourcing for Biochemistry
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F1,6BP and Antifoam (polypropylene glycol P 2000) were from Sigma-Aldrich Chemie GmbH (Munich, Germany), IPTG was from PEQLAB Biotechnologie GmbH (Erlangen, Germany), Bradford solution was from Biorad and sodium dodecyl sulfate from SERVA Electrophoresis GmbH (Heidelberg, Germany). All other chemical were either from Carl Roth GmbH (Karlsruhe, Germany), Fluka (Taufkirchen, Germany), or Merck KGaA (Darmstadt, Germany) and were of the highest available purity. The crude glycerol was delivered from a biodiesel producer (ADM Hamburg, Germany) and contained 83.20% glycerol, 10.4% water, 6.4% NaCl, with less than 0.01% methanol, and no detectable traces of other organic compounds (according to supplier’s data sheet). All restriction enzymes used were from New England Biolabs (Frankfurt, Germany). The Pwo DNA Polymerase for gene amplification was from Roche Applied Science (Mannheim, Germany), Taq DNA polymerase used for screening-PCR was from Genaxxon Bioscience GmbH (Ulm, Germany).
6
Prenatal PAH Exposure Protocol
7
Aerobic Thermophilic Cultivation of GXG-5 Cells
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The GXG-5 cells were inoculated into 100 mL of seed medium in 500 mL flask and aerobically cultured at 50 °C for 12 h with shaking at 200 rpm. The seed culture (200 mL) was inoculated into 7 L initial fermentation medium in a 10 L fermentor (BLBIO, China) to start the cultivation at 50 °C. The pH was automatically controlled at 7.0 ± 0.1 by adding 2 N NaOH and/or 2 N HCl. The aeration rate was maintained at 0.5 vvm and the agitation speed was gradually increased from 200 to 300 rpm to increase dissolved oxygen. Besides, 150 μL antifoam (Sigma, USA) was added at the beginning of fermentation to control the formation of foam.
8
Protein Purification and Enrichment Protocol
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250 μl of 10% brain homogenate is digested at 37°C for 24 hours with 250 μl of 200 μg/ml proteinase K solution. (Roche Diagnostics, Mannheim, Germany). To remove PK from the sample prior to PMCA, the PK digested brain homogenate is incubated at 37°C for 1 hour with 1 μl benzonase (MilliporeSigma, Burlington, MA). The sample is then incubated at room temperature for one hour with 250 μl of sarkosyl solution (20% N-lauroylsarcosine in 10 mM Tris Buffer pH 7.5), 1 μl DL-dithiothreitol 250 mM (Sigma-Aldrich, Burlington, MA), and 1 μl of Antifoam (Sigma-Aldrich, Burlington, MA). After incubation, the sample is centrifuged at 10,000 x g for 30 minutes, the pellet is discarded, and the supernatant centrifuged at 100,000 x g for 1 hour. The supernatant is discarded, the pellet resuspended in DPBS (Corning, Corning, NY) and centrifuged at 100,000 x g for 1 hour. The supernatant is discarded, the pellet resuspended in 0.1% sarkosyl solution (0.1% N-lauroylsarcosine in DPBS) and stored at -80°C.
9
Efficient DNA Extraction from Shark Tissues
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Elephant shark tissue samples were sourced as by-product of deceased animals harvested from commercial fishing in the Otago coastal region. As such, no animal ethics permission was applicable in this circumstance. No animal experimentation or manipulation was undertaken as defined by the Animal Welfare Act (2009, New Zealand), or according to guidelines issued by the New Zealand National Animal Ethics Advisory Committee (NAEAC, Occasional Paper No 2, 2009, ISBN 978-0-478-33858-4).
DNA was purified using a modified magnetic bead approach
44 (link). Briefly, cells were first homogenised in “GITC” lysis buffer (4 M Guanidine thiocyanate, Sigma G6639; 50 mM Tris, Thermo 15568-025; 20 mM EDTA; Thermo 15575-020; 2% Sarkosyl, Sigma L9150-50G; 0.1% Antifoam, Sigma A8311-50ML), and this lysate mixture was then combined with TE-diluted Sera-Mag Magnetic SpeedBeads (GE Healthcare, GEHE45152105050250) and isopropanol in a volumetric ratio of 2:3:4, respectively. Following capture with a neodymium magnet, beads were washed once with isopropanol, twice with 70% ethanol and resuspended in filter-sterile milliQ water.
DNA was purified using a modified magnetic bead approach
44 (link). Briefly, cells were first homogenised in “GITC” lysis buffer (4 M Guanidine thiocyanate, Sigma G6639; 50 mM Tris, Thermo 15568-025; 20 mM EDTA; Thermo 15575-020; 2% Sarkosyl, Sigma L9150-50G; 0.1% Antifoam, Sigma A8311-50ML), and this lysate mixture was then combined with TE-diluted Sera-Mag Magnetic SpeedBeads (GE Healthcare, GEHE45152105050250) and isopropanol in a volumetric ratio of 2:3:4, respectively. Following capture with a neodymium magnet, beads were washed once with isopropanol, twice with 70% ethanol and resuspended in filter-sterile milliQ water.
10
Optimized Cultivation of Pseudomonas putida KT2440
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The precultures of Pseudomonas putida KT2440 were used to inoculate a 7.0-L bioreactor (Biostat A, Sartorius, Germany) so that the initial OD600 was 0.1. The inoculation size was 10%. All cultivations were carried out at 30 °C. The temperature was maintained by a thermostatic jacket. The pH of each culture was maintained at 7.0 through the modulated addition of 1 N NaOH and 1 N HCl. The dissolved oxygen was monitored during the entire cycle with an O2 electrode (InPro 6800, Mettler Toledo GmbH, Greifensee, Switzerland). The agitation rate from 300 rpm to 1000 rpm was adjusted automatically to maintain 50% air saturation. The total fermentation time was 48 h. An antifoam solution (Sigma Aldrich, St. Louis, MO, USA) was used in response to the antifoam controller.
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