Separating the Supernatant and Solid Phases of a Sample by Microcentrifugation
Biochemistry, Molecular Biology, and Cell Biology Protocols >> Separating the Supernatant and Solid Phases of a Sample by Microcentrifugation
Perform microcentrifugation as described below, and when completed, carefully pipet the top of the liquid solution (the supernatant) into a separate tube. The pellet can be resuspended in a buffer of choice and analyzed.
A microcentrifuge usually has a maximum speed of about 13,000 to 14,000 rpm (revolutions per minute). The following guidelines refer to the 1.5 ml microcentrifuge tubes.
General precautions and principles when performing microcentrifugation:
- Read the manual thoroughly, and have it in a place readily accessible.
- You usually need to plug the microcentrifuge into a plug and turn on an on/off switch (not the same one as starting the spin) before the top cover can be opened.
- Ask someone who knows how to use the microcentrifuge to show you how to use it and perhaps watch you use it once. Make notes when watching if required.
- Mix solutions in a microcentrifuge tube by pipeting up and down the new contents into the old contents.
- Remember to always use a balance microcentrifuge tube containing the same amount of liquid as the sample, or to use two tubes with the same amount of each sample. If the liquid is more viscous or heavy, use a similarly viscous solution in the balance microcentrifuge tube. Place the balance tube at exactly the opposite end of the sample tube.
- Remember to place the lid on (screw it on reasonably tightly if there is a screw), before closing the top cover.
- Besides regular spins with regular time intervals, it is usually possible to do a quick spin. For quick spin, press the quick spin button for however long you would like to spin for. This is especially useful for accumulating all the liquid at the bottom of the tube. You may want to gently mix with your finger after taking the tube out of the centrifuge.
- To be consistent in case you ever want to know where a certain invisible pellet is, it is recommended to place the microcentrifuge tube with the notch perpendicular to the perimeter of the rotor facing out. That way, the pellet will always be at the same location.
- To separate the supernatant from a pellet, pipet directly from the bottom of the tube to remove the supernatant. If resuspending the pellet, pipet carefully without making any bubbles.
- For 0.5 ml microcentrifuge tubes, it may be desirable to place it into a 1.5 ml tube with the cap of the 1.5 ml tube cut off, and then placing the entire assembly into the microcentrifuge.