Swinging-Bucket Rotors

Basically, rotors come in four varieties fixed-angle rotors, vertical tube rotors, swinging-bucket rotors, and zonal rotors. The first three rotors are discussed below. Zonal rotors will be discussed In a later section. 

Fig. 11. (a) Diffeiential centrifugation (pelleting), where time 1 < time 2 < time 3 < time, (b) Rate 2onal separation in a swinging-bucket rotor, where tube 1 represents the density gradient solution, tube 2 the sample plus the gradient, and tube 3 the separation of sample particles under a centrifugal force, where... 

Add 30 mL of 15-50% (w/w) gradient solution to the ultracentrifuge tubes of the swinging bucket rotor (max vol 40 mL). Sucrose concentrations increase from the top to the bottom of the tube. 

Step 1. Load a disposable mini-chromatography col-umn with Sephadex G-50 (fine) dextran that was previously permitted to equilibrate with 5 volumes of the desired new buffer. Allow all the liquid to drain under the earth s gravitation to leave a 1 mL bed volume. Transfer this spin-column to an appropriately sized centrifuge tube and spin at 100 X g (calculated for the radial distance to the tip of the column itself, and not the bottom of the centrifuge tube) for 2 min in a swinging bucket rotor". The volume of the now semi-dry gel bed will be reduced by 25-40%, and the media will appear as a gel mass that may even break away from the walls of the chromatography tube. (This is normal and will not affect performance.)... 

The tubes of the swinging bucket rotor type are accommodated in a pivoted bucket moving from vertical to horizontal position during acceleration under the influence of the centrifugal field. The sedimentation path length is lowest in vertical rotors and highest in swinging-bucket rotors. 

Fig. 5.2. rpm-g graph for fixed-angle (type 45 Ti) and swinging-bucket (SW 28) Beckman Coulter ultracentrifuge rotor... 

Most laboratories have a standard low-speed centrifuge used for routine sedimentation of relatively heavy particles. The common centrifuge has a maximum speed in the range of 4000 to 5000 rpm, with RCF values up to 3000 X g. These instruments usually operate at room temperature with no means of temperature control of the samples. Two types of rotors, fixed angle and swinging bucket, may be used in the instrument. Centrifuge tubes or bottles that contain 12 or 50 mL of sample are commonly used. Low-speed... 

Rotors for a high-speed centrifuge. A Fixed angle B swinging bucket C vertical. Courtesy of Beckman Instruments, Inc. 

Operation of a swinging-bucket rotor. A Loading of sample. B Sample at start of centrifugation. C Sample during centrifugation separates into two components. D Rotor at rest. 

Tabletop centrifuge and appropriate swinging bucket rotor Vacuum filtering apparatus and Whatman filter no. 42 paper 30- to 50-ml tube with cap Shaking water bath or ultrasonic bath, 60°C...

Pellet cells for 5 min in two 50 mL Falcon tubes (e.g., 2000 rpm in a Beckman J-6M swinging bucket centrifuge with Beckman JS-4.2 rotor). (We use sterile Falcon tubes (BD Biosciences, San Jose, CA, cat. 352070) to minimize the chance of nuclease contamination.) The supernatant is removed. Optionally, one pellet can be resuspended in equal volume of LB (1% Bacto Tryptone, 0.5% yeast extract and 0.5% NaCl in water) and 60% glycerol, divided into multiple fractions and stored at —80 °C for future use. The other pellet is resuspended in 25 mL at 37 °C LB, then add into 2 L LB containing 2 mL of 50 mg/mL Carbenicillin and 2 mL of 10 mg/mL Chloramphenicol. Grow at 37 °C until the ODgoo is close to 1,... 

M., A one-step separation of human serum high density lipoproteins 2 and 3 by rate-zonal density gradient ultracentrifugation in a swinging bucket rotor. J, Lipid Res. 23, 1342-1353 (1982). 

Desk top clinical centrifuges (swinging bucket rotor)... 

Light scattering cells were of the type designed by Dandliker and Kraut (II) to permit the solution to be centrifuged in the cells for removal of dust. The cells were floated in sulfuric acid and centrifuged for 4—6 hours at 9000 r.p.m. in a Servall centrifuge equipped with a swinging bucket rotor. Cells suitable for use were selected from a hand-... 

The ground homogenate should be centrifuged at low speed (2500 rpm) for 5 min in a swinging-bucket rotor at 4°. The resulting pellet is discarded. Repetition of this low-speed spin (2500 rpm) 3 to 5 times ensures that most cellular contaminants and nuclei are pelleted and eliminated. Because the low-speed pellets contain nuclei, it is sometimes desirable to save these pellets for nuclear DNA preparations. 

Cap the tube with the Centricon retentate cup, then backspin the retentate (about 40 /zl) into the cup in a swinging-bucket rotor by bringing the speed to 1000 rpm and then braking immediately. The retentate contains the biotinylated dsDNA for use in the capture step below. 

Remove the filtrate cup and backspin the retentate into the retentate cup in a swinging-bucket rotor by raising the speed to 1000 rpm and braking immediately. 

Centrifuge with swinging bucket rotor and sealed microtiter plate carriers... 

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