Exercise 14.10 - Sucrose Density Fractionation

LEVEL II

Figure 14.2 Sucrose gradient distribution of RNA

Materials

• 10% and 40% (w/v) Sucrose 8
  
• 0.02 M sodium acetate, pH 5.1, containing 0.1 M NaCl and 1 mM
  
• EDTA
  
• RNA
  
• Ultracentrifuge, swinging bucket rotor and tubes
  
• Centrifuge tube fractionating device
  
• UV spectrophotometer

Procedure

1. 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 functioning.

   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 10% sucrose.

   e. Allow the flow to continue until all of the sucrose enters the centrifuge tube.

2. 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

3. 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.

4. 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).

5. 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 order.

   c. Push down on the centrifuge tube (Gently!) in order to puncture the bottom of the tube and immediately begin to collect the effluent.

   d. Count the drops that fall from the device, and place 40 drops into each tube.

6. Using a UV spectrophotometer 11 and microcuvettes, read the A of each fraction. Calculate the amount of RNA in each fraction.

7. 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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