The C. elegans liquid culture was chilled on ice for 30 min to allow the animals to settle. The culture supernatant was collected and cleared by centrifugation at 15,000 rpm for 30 min (JLA 16.250, Beckman coulter). Orsay virus in the supernatant was pelleted by centrifugation at 31,000 rpm for 2 h (SW32Ti rotor, Beckman coulter). The pellet was resuspended in 2 ml of CsCl gradient buffer (20 mM Tris–HCl pH7.8, 0.1% 2-Mercaptoethanol) at 4 °C overnight. The 2 ml of the virus suspension was applied to a 20–40% (weight/weight) CsCl linear gradient and centrifuged at 29,100 rpm (SW41Ti rotor, Beckman coulter) at 4 °C for 16 h. The 1 ml fractions were collected using a peristaltic fraction maker (Labconco Auto Dens-flow) linked to a Bio-rad fraction collector. The refractive index of each CsCl density fraction was measured and converted to the corresponding density (Fasman, 1976 ). To concentrate the virus, each fraction was mixed with 4 ml CsCl gradient buffer and centrifuged at 37,000 rpm (SW55Ti, Beckman coulter) for 2 h to pellet the virus. The pellet from each fraction was resuspended in 100 μl of CsCl gradient buffer.
Jla 16
Manufactured by Beckman Coulter
The JLA 16.250 is a high-performance centrifuge designed for a variety of laboratory applications. It features a maximum speed of 16,250 rpm and can generate a maximum relative centrifugal force of 30,228 x g. The centrifuge accommodates a range of rotor types and sample volumes, making it a versatile tool for various laboratory workflows.
Automatically generated - may contain errors
2 protocols using jla 16
1
Orsay Virus Purification from C. elegans
2
Bacterial Growth and Harvesting for RNA Analysis
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Bacteria were grown either at 37 °C (E. coli BW25113 derivatives) or 18 °C (E. coli ΔbipA CFT073 derivatives) up to OD600 of 0.5 in 50 mL of Neidhardt MOPS minimal medium [80] (link) supplemented with 0.1% casamino acids (w/v), 0.5% glycerol (w/v) and 100 μg/mL carbenicillin and l -arabinose was added to a final concentration of 0.2% (w/v). Cultures grown at 37 °C were harvested 10 min after induction by pouring them into precooled centrifuge bottles containing 100 g of crushed ice and centrifuged at 10,000 rpm for 10 min at 4 °C (Beckman JLA16.250 rotor). Cultures grown at 18 °C were harvested 5 h after induction by collecting into precooled centrifuge bottles and pelleting at 10,000 rpm for 10 min at 4 °C (Beckman JA25.50 rotor). Lysates were prepared the same as for polysome profiling of E. coli (see below).
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