Exercise 14.10 - Sucrose Density Fractionation
Figure 14.2 Sucrose gradient distribution of RNA
- 10% and 40% (w/v) Sucrose 8
- 0.02 M sodium acetate, pH 5.1, containing 0.1 M NaCl and 1 mM
- Ultracentrifuge, swinging bucket rotor and tubes
- Centrifuge tube fractionating device
- UV spectrophotometer
- Refer to Chapter Three for details of fractionation, and
form a 10-40% linear sucrose gradient in a nitrocellulose tube.
a. Close all valves on the gradient device.
b. Place exactly 15 ml.9
of 40% sucrose in the right chamber and 15 ml. of 10% sucrose in
the left chamber of the gradient device.
c. Open the flow from the right chamber to the centrifuge tube.
Be sure there is a tube in place, that the flow is directed down
the inside of the tube and that the magnetic stirrer is
d. Immediately open the valve on the left chamber and insure
that sucrose is
flowing from left to right, thereby diluting the 40% sucrose with
e. Allow the flow to continue until all of the sucrose enters
the centrifuge tube.
- Dissolve the RNA in 0.02 M sodium acetate solution to yield
a final concentration of 250 µg/ml. The low pH of the solution
helps to inhibit RNAse, while the salts will keep the RNA from
forming large polymeric aggregates. 10
- Carefully layer 2.0 ml of the dissolved RNA onto the top of
a sucrose gradient. This should be done by slowly allowing the
solution to run down the side of the tube and onto the gradient.
Be careful not to disturb the gradient.
- Load the ultracentrifuge with the prepared tubes and
centrifuge for the equivalent of 18,700 RPM for a Beckman SW27
rotor, for 20 hours at 4° C (Refer to Appendix F).
- At the completion of centrifugation, remove the tubes and
fractionate the contents into 1.0 ml fractions.
a. Insert the nitrocellulose centrifuge tube into the plexiglass
holder, but be careful NOT to puncture the bottom.
b. Have a series of 30-35 test tubes ready to accept the
effluent from the tubes. Each tube should be labeled and kept in
c. Push down on the centrifuge tube (Gently!) in order to
puncture the bottom of the tube and immediately begin to collect
d. Count the drops that fall from the device, and place 40 drops
into each tube.
- Using a UV spectrophotometer 11
and microcuvettes, read the A
of each fraction. Calculate the amount of RNA in each fraction.
- Plot the concentration of RNA in each fraction against the
fraction number. Based on the density of sucrose in each
fraction, compute the density of the RNA in that fraction. Based
on the relative size (greater density) of the RNA, determine the
nature of the fractions (i.e. rRNA, tRNA).
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Cell Biology Laboratory Manual
Dr. William H. Heidcamp, Biology Department, Gustavus Adolphus College,
St. Peter, MN 56082 -- firstname.lastname@example.org