Swinging-bucket centrifuge

Because various swinging bucket centrifuges behave differently with respect to acceleration and braking, some optimization of centrifugation conditions may be advisable. This can be readily accomplished by UV and visible spectrophotometry if one uses BSA (or some other inexpensive protein sample) along with a small amount of dichromate as a yellow colored metal ion species. 

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,... 

Swinging-bucket centrifuge (with adaptors for spinning 50-ml tubes). Microcentrifuge. 

Swinging bucket centrifuge with adaptor for 50-ml tubes. 

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... 

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. 

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...

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. 

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

The procedure can be further accelerated for the separation of components of very different densities using step gradients in a swinging-bucket rotor. Consider, for example, the separation of RNA and DNA. To separate these, the centrifuge tube is first half-filled with a CsCl2 solution of density 1.76 103 kg m-3, and this is overlaid with a less dense solution (ca. 1.65 103 kg m 3). Since DNA (ca. 

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