ROTOR PATHLENGTH (Rmax-Rmin) Figure 4.2 Effect of rotor angle on centrifugation experiments. Reproduced with permission of Kendro Laboratory Products. Artwork by Murray Levine. e the radius of a swinging bucket rotor is the distance between fu. enter of the rotor and the bottom of the bucket when it is horizontal(Figure 4.2a). The greater the rotor radius, the greater is the g force. The distance between the center of a fixed angle rotor and the bottom of the tube cavity determines the radius of a fixed-angle rotor(Figure 4.2b). Again the g force increases directly as the radius increases The greater the rotor angle, the greater is the distance the sample must travel before it pellets( Figure 4.2b ). This travel dis- tance also affects the shape of a density gradient(Figure 4.3) The k-factor of a fixed-angle rotor provides a method to predict the required time of centrifugation for different fixed-angle rotors How to Properly Use and Maintain Laboratory Equipment
How to Properly Use and Maintain Laboratory Equipment 59 The radius of a swinging bucket rotor is the distance between the center of the rotor and the bottom of the bucket when it is fully horizontal (Figure 4.2a). The greater the rotor radius, the greater is the g force. The distance between the center of a fixed angle rotor and the bottom of the tube cavity determines the radius of a fixed-angle rotor (Figure 4.2b). Again the g force increases directly as the radius increases. The greater the rotor angle, the greater is the distance the sample must travel before it pellets (Figure 4.2b). This travel distance also affects the shape of a density gradient (Figure 4.3). The k-factor of a fixed-angle rotor provides a method to predict the required time of centrifugation for different fixed-angle rotors. Rmin Rmin Rmax Rmin a b c ROTOR PATHLENGTH (Rmax–Rmin) Figure 4.2 Effect of rotor angle on centrifugation experiments. Reproduced with permission of Kendro Laboratory Products. Artwork by Murray Levine
Figure 4.3 Effect of rotor angle on gradient formation Reproduced with permis- sion of amersham pharmacia 409 Rotor 40.2 E9E995苏 Rotor 60.5 140→ 8 Rotor 30.2 1.10 Density g/ml The k-factor is a measure of pelleting efficiency; rotors with smaller k-factors(smaller fixed angles or vertical angles)pellet more efficiently, requiring shorter run times. The k-factor can also calculate the time required to generate a gradient when switching between different rotors the k-factor can be determined by k Equation(2)uses the k-factor to predict the time required for centrifugation for different fixed-angle rotors T T2 Ti is the run time in minutes for the established protocol. First calculate the k, factor at the appropriate speed for the rotor that is referenced. Next, calculate the k2 factor at the chosen speed for your rotor. Finally, solve for T2. This strategy is not appropriate to Troutman et al
60 Troutman et al. The k-factor is a measure of pelleting efficiency; rotors with smaller k-factors (smaller fixed angles or vertical angles) pellet more efficiently, requiring shorter run times. The k-factor can also calculate the time required to generate a gradient when switching between different rotors. The k-factor can be determined by (1) Equation (2) uses the k-factor to predict the time required for centrifugation for different fixed-angle rotors: (2) T1 is the run time in minutes for the established protocol. First, calculate the k1 factor at the appropriate speed for the rotor that is referenced. Next, calculate the k2 factor at the chosen speed for your rotor. Finally, solve for T2. This strategy is not appropriate to T k T k 1 1 2 2 = k rpm = ¥ ( ) Ê Ë Á ˆ ¯ ˜ ( ) 2 53 1011 2 . ln r r max min 40° Rotor 40.2 Rotor 60.5 Rotor 30.2 1.00 1.10 1.20 Density g/ml 14° 23.5° 8 6 4 2 6 4 2 6 4 2 Distance from the meniscus (cm) Figure 4.3 Effect of rotor angle on gradient formation. Reproduced with permission of Amersham Pharmacia Biotech
convert protocols between rotor types(fixed-angle, horizontal nd vertical) Should Flamable, Explosive, or Biohazardous Materials Be Centrifuged in Standard Centrifugation Equipment? Centrifuge manufacturers strongly recommend that standard laboratory centrifuges should not be exposed to any materials capable of producing flammable or explosive vapors, or extreme exothermic reactions. Specialized equipment exists for centrifug- ing dangerous substances. Which Centrifuge Tube Is Appropriate for Your Application? a broken or leaking sample container can seriously damage a centrifuge by knocking the rotor out of balance or exposing mechanical and electrical components to harsh chemicals Damage can occur at any speed. Use only the tubes that are manufacturer to assess compatabllfi. If unsure, contact the tube recommended for centrifugation us With the trend toward smaller sample size and greater througl put, microplates have become very popular. Other protocols call for vials and slides. Never attempt to create your own adapter for these containers ask the rotor manufacturer about the availabil ity of specialized equipment Correct tube fit is critical, especially at higher g forces. Tubes or containers that are too large can get trapped in rotors, while tubes that fit loosely can leak or break. Never use homemade adapters While a broken tube doesn't sound costly, poorly fitted containers can lead to costly repairs. g Force Many tubes are not suitable for high stress centrifugation.When in doubt about g force limitations, contact the tube's manufac turer. If this isn't feasible, you can test the tube by filling it with water, centrifuging at low rpm's, and inspecting the tube for damage or indications of stress while slowly increasing the speed Chemical Compatibility Confirm the tube's resistance with the manufacturer. Contain ers that are not resistant to the sample might survive one or more centrifugations but will surely be weakened. Chemically resistant containers should always be inspected for signs of stress before using them. Repeated centrifugation can damage any container. How to Properly Use and Maintain Laboratory Equipment 6
convert protocols between rotor types (fixed-angle, horizontal, and vertical). Should Flamables, Explosive, or Biohazardous Materials Be Centrifuged in Standard Centrifugation Equipment? Centrifuge manufacturers strongly recommend that standard laboratory centrifuges should not be exposed to any materials capable of producing flammable or explosive vapors, or extreme exothermic reactions. Specialized equipment exists for centrifuging dangerous substances. Which Centrifuge Tube Is Appropriate for Your Application? A broken or leaking sample container can seriously damage a centrifuge by knocking the rotor out of balance or exposing mechanical and electrical components to harsh chemicals. Damage can occur at any speed. Use only the tubes that are recommended for centrifugation use. If unsure, contact the tube manufacturer to assess compatability. With the trend toward smaller sample size and greater throughput, microplates have become very popular. Other protocols call for vials and slides. Never attempt to create your own adapter for these containers; ask the rotor manufacturer about the availability of specialized equipment. Fit Correct tube fit is critical, especially at higher g forces. Tubes or containers that are too large can get trapped in rotors, while tubes that fit loosely can leak or break. Never use homemade adapters. While a broken tube doesn’t sound costly, poorly fitted containers can lead to costly repairs. g Force Many tubes are not suitable for high stress centrifugation.When in doubt about g force limitations, contact the tube’s manufacturer. If this isn’t feasible, you can test the tube by filling it with water, centrifuging at low rpm’s, and inspecting the tube for damage or indications of stress while slowly increasing the speed. Chemical Compatibility Confirm the tube’s resistance with the manufacturer. Containers that are not resistant to the sample might survive one or more centrifugations but will surely be weakened. Chemically resistant containers should always be inspected for signs of stress before using them. Repeated centrifugation can damage any container. How to Properly Use and Maintain Laboratory Equipment 61
A Checklist for Centrifuge Use Inspect the centrifuge for frost on the inside chamber. Accu- mulated frost must be removed because it can prevent proper temperature control. Previous spills should also be cleaned before tarting the centrifuge If your instrument uses rotor identification codes, does your instrument have the appropriate software to recognize and operate your rotor? Don't apply the identification code of rotor for a second rotor that does not possess it's own code Manually confirm the speed limitations of your rotor if identifi cation codes aren't relevant Inspect the rotor for signs of corrosion and wear-and-tear If you ee any pitting or stress marks in the rotor cavities, do not use the rotor. If it is difficult to lock the rotor lid down or lock the rotor o the centrifuge, don't use the rotor. Check that all O-rings on he rotor and sample holders are present, clean, in good physical condition, and well lubricated. Many fixed-angle rotors have a cover O-ring, while many rotors that get locked to the drive hav a drive spindle O-ring. If you have concerns about the rotor's con- dition, dont use it. Request an inspection from the manufacturer All the buckets and/or carriers within a swinging bucket rotor must be in place, even when these positions are empty. Utilize the proper adaptors and tubes, as described above. Balance your tubes or bottles. Refer to the manufacturer's instructions for balance to erance, which vary with different rotors. Place the rotor onto the drive shaft and check that it is seated properly. Many rotors must be secured to the drive. gently try to lift the rotor off the drive as a final check that the rotor is properly installed Begin centrifugation. Even though most imbalances occur at lower speed, monitor the centrifuge until it approaches final speed. If an imbalance occurs, reinspect the balancing of the tubes and the placement of the rotor Should the brake Be applied, and If so, to What Degree? If a brake is turned completely off, it could take hours for the rotor to stop, depending on the top speed and instrument condi tions (i.e, vacuum run). The stiffer the brake setting, the greater the jolt to the sample, so take note that the default setting of most instruments is the hardest, quickest brake rate. A reduced brake rate is recommended when separating samples of similar densi- ties, when high resolution gradients and layers are required, and when fluffy, noncompacted pellets are produced. The degree of jolting, the braking technique, and the terminology varies among Troutman et al
A Checklist for Centrifuge Use Inspect the centrifuge for frost on the inside chamber. Accumulated frost must be removed because it can prevent proper temperature control. Previous spills should also be cleaned before starting the centrifuge. If your instrument uses rotor identification codes, does your instrument have the appropriate software to recognize and operate your rotor? Don’t apply the identification code of one rotor for a second rotor that does not possess it’s own code. Manually confirm the speed limitations of your rotor if identifi- cation codes aren’t relevant. Inspect the rotor for signs of corrosion and wear-and-tear. If you see any pitting or stress marks in the rotor cavities, do not use the rotor. If it is difficult to lock the rotor lid down or lock the rotor to the centrifuge, don’t use the rotor. Check that all O-rings on the rotor and sample holders are present, clean, in good physical condition, and well lubricated. Many fixed-angle rotors have a cover O-ring, while many rotors that get locked to the drive have a drive spindle O-ring. If you have concerns about the rotor’s condition, don’t use it. Request an inspection from the manufacturer. All the buckets and/or carriers within a swinging bucket rotor must be in place, even when these positions are empty. Utilize the proper adaptors and tubes, as described above. Balance your tubes or bottles. Refer to the manufacturer’s instructions for balance tolerance, which vary with different rotors. Place the rotor onto the drive shaft and check that it is seated properly. Many rotors must be secured to the drive. Gently try to lift the rotor off the drive as a final check that the rotor is properly installed. Begin centrifugation. Even though most imbalances occur at lower speed, monitor the centrifuge until it approaches final speed. If an imbalance occurs, reinspect the balancing of the tubes and the placement of the rotor. Should the Brake Be Applied, and If So, to What Degree? If a brake is turned completely off, it could take hours for the rotor to stop, depending on the top speed and instrument conditions (i.e., vacuum run). The stiffer the brake setting, the greater the jolt to the sample, so take note that the default setting of most instruments is the hardest, quickest brake rate. A reduced brake rate is recommended when separating samples of similar densities, when high resolution gradients and layers are required, and when fluffy, noncompacted pellets are produced. The degree of jolting, the braking technique, and the terminology varies among 62 Troutman et al
manufacturers, so consult your operating manual or contact the manufacturer to discuss the most appropriate brake setting fo your application. [The reverse is true when looking at the decel eration. If you have an option as to where to control to(or from) 1000 to 1500rpm is recommended Centrifugation of DNA and RNA How Does a Deration Curve Affect Your Purification Strategy? Deration describes the situation where a rotor should not attain its maximum speed because a high-density solution is used in the separation. For example, centrifuged at high g force, dense solutions of CsCl will precipitate at a density of about 1.9g/ml,a situation that can blow out the bottom of the rotor The deration curve supplied by the rotor's manufacturer will indicate thos speeds that could cause centrifugation media to precipitate and potentially damage the rotor and or centrifuge(Figure 4.4) Is a Vertical Rotor the right Angle for You? Vertical rotors can purify DNa via cesium chloride centrifuga tion in three hours, as compared to the overnight runs using fixed angle rotors. Vertical rotors re-orient your sample(Figure 4.5),so there is the small possibility that the rna that pelleted against the outer wall of the tube will contaminate the dna as the gra dient re-orientes a near-vertical rotor from Beckman-Coulter eliminates this problem(Figure 4.6).The 9 angle of this rotor allows the RNa to pellet to the bottom of the tube without contaminating the dnA The closeness to vertical keeps centrifugation times short. Triton X-100 was applied in this near-vertical system to improve the sep- aration of RNA from plasmid DNA, although the impact of the detergent on the later applications of the DNa was not tested (Application Note A-1790A, Beckman-Coulter Corporation) Vertical rotors also allow for the tube to be pulled lled out straight without the worry of disrupting the gradient Fixed-angle rotors produce bands at an angle, requiring greater care when removing samples from the rotor What Can You do to Improve the Separation of supercoiled DNA from Relaxed Plasmid? Centrifugation at lower g force will increase the resolution of supercoiled and relaxed DNA. Apply a step-run gradient, where high speed establishes the gradient, followed by lower speeds and g forces to better separate supercoiled and relaxed DNA Rotor How to Properly Use and Maintain Laboratory Equipment 63
How to Properly Use and Maintain Laboratory Equipment 63 manufacturers, so consult your operating manual or contact the manufacturer to discuss the most appropriate brake setting for your application. [The reverse is true when looking at the deceleration.) If you have an option as to where to control to (or from), 1000 to 1500rpm is recommended. Centrifugation of DNA and RNA How Does a Deration Curve Affect Your Purification Strategy? Deration describes the situation where a rotor should not attain its maximum speed because a high-density solution is used in the separation. For example, centrifuged at high g force, dense solutions of CsCl will precipitate at a density of about 1.9 g/ml, a situation that can blow out the bottom of the rotor. The deration curve supplied by the rotor’s manufacturer will indicate those speeds that could cause centrifugation media to precipitate and potentially damage the rotor and or centrifuge (Figure 4.4). Is a Vertical Rotor the Right Angle for You? Vertical rotors can purify DNA via cesium chloride centrifugation in three hours, as compared to the overnight runs using fixedangle rotors. Vertical rotors re-orient your sample (Figure 4.5), so there is the small possibility that the RNA that pelleted against the outer wall of the tube will contaminate the DNA as the gradient re-orientes. A near-vertical rotor from Beckman-Coulter eliminates this problem (Figure 4.6).The 9° angle of this rotor allows the RNA to pellet to the bottom of the tube without contaminating the DNA. The closeness to vertical keeps centrifugation times short. Triton X-100 was applied in this near-vertical system to improve the separation of RNA from plasmid DNA, although the impact of the detergent on the later applications of the DNA was not tested (Application Note A-1790A, Beckman-Coulter Corporation). Vertical rotors also allow for the tube to be pulled out straight without the worry of disrupting the gradient. Fixed-angle rotors produce bands at an angle, requiring greater care when removing samples from the rotor. What Can You Do to Improve the Separation of Supercoiled DNA from Relaxed Plasmid? Centrifugation at lower g force will increase the resolution of supercoiled and relaxed DNA. Apply a step-run gradient, where high speed establishes the gradient, followed by lower speeds and g forces to better separate supercoiled and relaxed DNA. Rotor