Preparation of gradients for cells 

The most widely used method for producing discontinuous gradients is to start with the densest solution and layer solutions of successively lower densities on top using some form of pipette or syringe. 

Tilt the centrifuge tube (approx. 45); place the tip of the pipette or syringe against the wall of the tube, about 1 cm above the meniscus of the denser solution, and gently deliver a slow and steady stream of liquid. This allows the liquid to spread over the tube surface and minimizes any mixing due to a sudden increase in liquid flow. 

Once a steady flow is established keep the tip of the pipette or syringe just above the meniscus of the liquid and against the wall of the tube. From a pipette Use a rubber two- or three-valve pipette filler to aliquot and dispense the gradient solutions. Check that the release valve when pressed gently, allows the delivery of a slow and steady flow of liquid.

 Do not use a pipette filler that uses positive pressure to deliver the liquid, as a slow even flow is often difficult to attain. Always take up more of the gradient medium than is required as it is easier and more accurate to empty the pipette to a graduation mark than to try to empty it completely.