SUCROSE GRADIENT PROCEDURE

DENSITY GRADIENT CENTRIFUGATION-SUCROSE GRADIENT

AIM:

 To prepare a density gradient centrifugation for the separation of molecules from the sample solution

PRINCIPLE:

Sucrose density gradient ultracentrifugation is a powerful technique for fractionating macromolecules like DNA, RNA, and proteins. For this purpose, a sample containing a mixture of different size molecules is layered on the surface of a gradient whose density increases linearly from top to bottom. During centrifugation, different sized molecules sediment through the gradient at different rates. The rate of sedimentation depends, in addition to centrifugal force, on the size, shape, and density of the molecules, as well as on the density and viscosity of the gradient. In this way, molecules are separated by size with larger ones sedimenting towards the bottom and lighter ones remaining close to the top of the gradient. The method has been particularly successful in the size fractionation of large molecules.

MATERIALS REQUIRED:

-50%, 40%, 30%, and 25% (w/v) Sucrose solution

-Ultracentrifuge, Centrifuge tubes, Micropipette tips, Beakers

PROCEDURE:

1.      The gradient is prepared by layering progressively less dense sucrose solutions upon one another; therefore the first solution applied is the 50 % sucrose solution.

2.      Firstly a tube is held upright in a tube stand.

3.      Next a 200 μl pipettor tip is placed on the end of a 1000 μl pipettor tip.  Both snugly fitting tips are held steady by a clamp stand and the end of the 200 μl tip is allowed to make contact with the inside wall of the tube. 

4.      Now sucrose solutions can be placed inside the 1000 μl tip and gravity will feed the solutions into the tube slowly and steadily, starting with the 50 % solution.

5.      Once the 50 % solution has drained into the tube, the 40 % solution can be loaded into the 1000 μl tip which will then flow down the inside of the tube and layer on top of the 50 % solution. 

6.      This procedure is continued with the 30 % and 25 % respectively. 

7.      Once the sucrose gradient is poured discrete layers of sucrose is visible.

8.       Centrifugation should begin as soon as possible.

9.      However all centrifugation procedures require a balanced rotor therefore another tube containing precisely the same mass must be generated. 

10.  In practice this means 2 gradients must be prepared although the second gradient need not contain an experimental sample but could contain 0.5 ml water in place of the 0.5 ml of sample.  

11.  The tubes are centrifuged in a swinging bucket type rotor at 37 500 rpm for 16 hours 30 minutes at 4^oC.

12.  Immediately after the run the tube should be removed from the rotor, taking great care not to disturb the layers of sucrose.

13.  For fraction collection the tube should be held steady and upright by a clamp stand.  A tiny hole should be introduced into the very bottom of the tube using a fine needle.  The hole should be just big enough to allow the sucrose solution to drip out at approximately 1 drop per second. 

14.  Fractions of equal volume are then collected in eppendorf tubes below the pierced hole.  The fractions can now be stored at – 80^oC.

RESULT:

When separating a sample, discrete layers were observed.

The diagram below demonstrates this process in the collection of different fraction.

Collecting fractions from sucrose density gradient