1. Attach capillary tubes to the ends of the tubing emerging from the gradient maker (our gradient maker can make 3 gradients simultaneously; even if you only want two, attach tubes to all the tubing). Be very careful not to break the capillary tubes; they are fairly fragile. Put 1, 2, or 3 ultracentrifuge tubes in a rack.
2. Close the mixer between the two sides of the maker. Add the higher-percentage sucrose solution to the non-outlet side of the maker (if making 2 gradients, use 16 ml/side for 16 ml gradients...3 gradients = 24 ml/side). Start the stirring motor, and add the lower-percentage sucrose solution to the outlet side. If you are fortunate, the sucrose will start flowing through the tubing. Great: pick lines that are flowing well, stick them in the ultracentrifuge tubes, and open the mixer between the two sucrose solutions (make sure you see high-concentration sucrose being whipped into the outlet side of the gradient maker). If you have unused lines (as when making only 2 gradients), raise the end of the unused tubing over the height of the sucrose reservoirs.
3. If the sucrose does not begin flowing through the tubing (this often occurs if the tubing is still moist from prior gradient-making runs), suction it along by attaching the suction bulb tubing to the end of the capillary tubes. As each one starts flowing, temporarily pause it by raising the end of the capillary tube over the height of the sucrose reservoirs. When all are flowing, lower into ultracentrifuge tubes and open mixer as above.
4. Keep an eye on the levels of the growing gradients. If the levels are uneven, adjust the relative heights of the ultracentrifuge tubes (i.e., lower the slower-forming gradient) to re-balance the levels.
5. When the gradients are completed, stop the stirrer and carefully remove the capillary tubes from the gradients. Remove the capillary tubes from the tubing, and put the ends of the tubing in a beaker. Run at least 3 volumes of distilled water through the gradinet maker, empty the collection beaker, and cover the gradient maker. Make sure you wipe up any sticky spots of sucrose from the surrounding area (including the floor).
6. Add a 1 ml 60% sucrose cushion to the bottom of the gradients by drawing 60% sucrose up in a syringe fitted with a long blunt-end needle. Gently insert the needle through each gradient to the bottom of the ultracentrifuge tube, and dispense 1 ml of sucrose. Carefully remove the needle.
7. Load sample on top of gradient with pipetman, balance tubes, and spin.
1. Flush pump tubing with distilled water, then run dry. Fill fraction collector with 35 microfuge tubes.
2. Carefully insert the long blunt-end needle into an ultracentrifuge tube containing a sucrose gradient, ensuring the needle rests at the bottom of the tube. Start the pump at 1 ml/min, and place the outlet for the fraction collecter over the first microfuge tube. Set the fraction size to 0.57 min (0.57 ml). When the first drop appears at the outlet, start the fraction collector.
3. After 18 min, check to make sure all sucrose has run into microfuge tubes (there should be 30 fractions). Flush the pump tubing with water for a few minutes, reload the fraction collector with fresh microfuge tubes, dry the pump tubing, and fractionate the next sucrose gradient.
4. When finished, flush tubing with at least 50 ml of distilled water and run dry.