16
SW 41 Ti Rotor
or from homogeneous solutions. But the total amount of CsCl in solu-
tion must be equivalent to a homogeneous solution corresponding to
the concentrations specified in Figure 3.) Figure 3 can also be used to
approximate the banding positions of sample particles. Curves not
shown in the figure may be interpolated.
ADJUSTING FILL VOLUMES
Figures 2 and 3 show that several fill volumes are possible in a tube.
If a thinwall tube is partially filled with gradient solution, float
mineral oil (or some other low-density, immiscible liquid) on top
of the tube contents to fill the tube to its maximum volume. (Do not
use an oil overlay in Ultra-Clear tubes.) Note that for a given CsCl
density, as the fill level decreases the maximum allowable speed
increases. Partial filling may be desirable when there is little sample
or when you wish to shorten the run time.
For example, a
quarter-filled
tube of 1.5-g/mL homogeneous CsCl
solution at 4°C may be centrifuged at 41 000 rpm (see Figure 2). The
segment of the 41 000-rpm curve (Figure 3) from the quarter-filled
line to the tube bottom represents this gradient. The same solution
in a
half-filled
tube may be centrifuged no faster than 33 000 rpm
(curves not shown in the figure may be interpolated), and 28 000 rpm
in a
three-quarter-filled
tube. A tube
full
of the 1.5-g/mL CsCl
solution may be centrifuged no faster than 25 000 rpm.
TYPICAL EXAMPLES FOR DETERMINING CsCl RUN PARAMETERS
Example A: Starting with a homogeneous CsCl solution density
of 1.6 g/mL and approximate particle buoyant
densities of 1.69 and 1.72 g/mL, at 20°C, where will
particles band at equilibrium?
1. In Figure 2, find the curve that corresponds to the required run
temperature (20°C) and fill volume (one-half full). The maximum
allowable rotor speed is determined from the point where this
curve intersects the homogeneous CsCl density (30 000 rpm).
2. In Figure 3, sketch in a horizontal line corresponding to each
particle's buoyant density.
3. Mark the point in the figure where each particle density intersects
the curve corresponding to the selected run speed and temperature.
Buffer
Gradient
