A Reliable, Fast, and Inexpensive Method of Preparing Sucrose Density Gradients Paul M. Taslimi and Demetre Kolokotronis Concordia University, 1455 DeMaisonneuve West, Montreal. PQ. Canada, H4GlMB The traditional method of preparing sucrose density gradients where an expensive gradient maker is not available. has been to introduce the layers of sucrose solutions-starting from the lowest density-with a pipet and allowing the solution to trickle down the side of a polyallomer tube. Each successively heavier layer displaces the lighter layers before it until the most dense solution is introduced. Alternatively the sucrose solutions can be introduced in order of decreasing density, but mixing can be a serious problem. In either case one must Bttempt the preparation several times before good gradients are obtained consistently. In the following method for the preparation of sucrose density grqdients, gradients of excellent quality are almost guaranteed at the first attempt. This method offers the advantages of minimum mixing and increased convenience in terms of speed and reproducibility. The basic apparatus consists of a 25- or 50-Lcalibrated glass capillary tube inserted into one of two holes made in a small rubber bung that fits closely onto the polyallomer tube in which the gradient is to be made (see figure). The size of the holes in t$ bung must be small enough to allow a close fit for the capdlary tube. The bung is then placed on top of the tube, and the capillary is pushed down into the tube until its end just touches the bottom of the tube (see figure). Care must be taken not to break the capillary by pushingtoo hard. ~ water, the cadlIf the hole is wetted sliehtlv with a d r o of lary will move quite e a h y . ~ e x ta, 3-t& disposable syringe is attached bv a 16-gauge needle to the end of the capillary tube via a small l e n i t h ( l cm) of rigid polyethylene tubing (id. -5 mm, 0.d. -2 mm) placed at the end of the capillary. A smalllength (5 em) of polyethylene capillary tubing is now inserted into the second hole in the rubber bung until one end protrudes about 1mm into the centrifuge tube-this is the air tube. The apparatus is now assembled and must be supported vertically (see figure). The exact xolumes of sucrose solutions are now placed onto 4-in. test tubes. The solutions are now removed one by one with a 9-in. Pasteur pipet, starting with the least dense, and poured into the syringe barrel. The first sucrose solution may have a bit of trouble going through because of air in the capillary, but tapping the plastic connecting tube will cause it to flow. If the gauge of the capillary tube is adequate, the
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Journal of Chemlcal Education
flow will be quite gentle. As the first sucrose solution is going into the tube, the next one should be removed, and, just as the first solution reaches the end of the barrel, the next sucrose solution is introduced into the barrel. It is crucial that the syringe barrel not empty completely at any time; otherwise, an air bubble will be introduced into the eradient when the next sucrose solution is introduced into the barrel, which may disturb it. After the last (and most dense) sucrose solution is introduced, it is allowed to run through completely. The gradient is now ready. The glass capillary tube should be extracted gently while holding the rubber bung firmly over the gradient tube. It is clear that the only disturbance experienced by the gradient is the removal of the capillary tube, which, if done carefully, affects the gradient minimally. This method is now used by third-year undergraduate students in our biochemistry laboratory with excellent results.
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Pasteur
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Assembly of me apparatus for the preparation of sucrose denshy gradients.
