As rotor slows down or stops, gravity causes the sediment to slide down the tube, usually a poorly packed pellet is formed. Vertical rotors are fairly specialized — their most common use is during ultracentrifugation for isopycnic separations , specifically for the banding of DNA in cesium chloride.
In this type of separation, the density range of the solution contains the same density as the particle of interest; thus the particles will orient within this portion of the gradient. Isopycnic separations are not dependent on the pathlength of the gradient but rather on run time, which must be sufficient for the particles to orient at the proper position within the gradient.
Vertical rotors have very low K factors typically in the range of 5—25 , indicating that the particle must only travel a short distance to pellet or in this case form a band ; therefore run time is minimized. Once it is determined that a vertical rotor is appropriate for the end-user application, volume and speed become the deciding factors for which rotor to use.
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The total mass of each tube should be as close as possible- this becomes increasingly important at very high rotor speeds. Balancing masses to the nearest 0. For example, do not balance a sample consisting of liquid with a higher or lower density than water with an equal volume of water. A little vibration is normal, but excessive amounts can mean danger.
First, double check that the tubes are correctly balanced. If this does not resolve the issue, do not operate the centrifuge until it has been serviced by the manufacturer or dealer. Prior to starting the centrifuge, it is necessary to load it correctly. Balancing the centrifuge prevents potential damage to the instrument, and is crucial for safe operation. There are two ways to balance three tubes.
The first option is to insert three sample tubes next to each other, and create three balance tubes to be situated directly across from the sample tubes. To balance five tubes, create one balance tube and place two sets of three tubes across from each other. To balance seven tubes, create one balance tube and place two sets of four tubes across from each other. A few simple steps can keep a centrifuge functioning properly and reduce the risk of damage or injury.
Regularly clean the centrifuge with neutral cleaning solutions alcohol or alcohol-based disinfectant applied with a soft cloth to rotors and accessories. Rotors made of metal, such as steel and aluminum, have a high density and high thermal conductivity. They transfer heat efficiently and get chilled quickly. On the contrary, materials like polymers and carbon fibers are heat insulators and maintain a constant temperature.
The shape of the rotor determines the airflow within a centrifuge, similar to how the blades of an electric fan influence airflow in and out of the machine. Optimizing the airflow within a centrifuge through rotor shape is essential to maintaining the temperature. The speed of rotation is directly proportional to the rise in temperature—at higher speeds, more heat is generated. It is important to understand the maximum speed of the centrifuge and the range of speeds that maintain a temperature range that will not change the outcome of the experiment.
Usually, this information will be provided by the manufacturer in the equipment manual to help the user understand the limitations and work around it. During centrifugation, separation of the sample components continues during the deceleration phase. Many centrifuges offer the option to control deceleration settings brakes to bring it to a stop faster, but when do we really need this option, and what is the effect on sample outcomes?
Braking can be particularly useful during centrifugations involving nucleic acid extractions or bacterial cell pelleting, which are not affected by sudden stopping. However, for experiments that are more sensitive to abrupt deceleration, such as isolation of peripheral blood mononuclear cells and gradient centrifugations, braking can cause separated layers to remix.
In such cases, it is more suitable to turn off the brake so that deceleration is gradual and does not disturb the gradients. Some centrifuges provide a range of deceleration settings. This can be useful when spinning mammalian cells, which are sensitive to sudden decelerations but, at the same time, require some deceleration to minimize the time taken by the centrifuge to come to a stop.
One of the most common applications of centrifugation is to pellet samples, such as bacterial cells, mammalian cells, or nucleic acids. While using a fixed-angle rotor, the angle of the rotor determines the position of the pellet.
To ensure you always know where your pellet is, a good lab practice is to always spin the tubes with the lid hinges placed in the same orientation for example, lid hinges facing outwards. This is especially useful to prevent sample loss during DNA extractions when the pellet is nearly invisible following the ethanol wash step.
In the case of swing-bucket rotors, the pellets are formed mostly at the bottom of the tube. Overall, I hope reading this article helps you to work more comfortably and confidently with centrifuges. If you have seen or lived through a centrifuge disaster, go ahead and share your story along with any tips and tricks to ensure happy spinning in the comments below.
May the force be with you! Has this helped you?
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