Tabletop Rotors and Tubes

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3 Using Tubes and Accessories This section contains general instructions for filling and capping the labware used in Beckman Coulter preparative rotors, for selecting and using the appropriate accessories, and for recovering samples after a run. Individual rotor manuals provide specific instructions on tubes and accessories that can be used in a particular rotor. 1 Rotor use instructions are in Section 4. A table of chemical resistances is in Appendix A of this manual. Reference information on some commonly used gradient materials is in Appendix C. GRADIENT PREPARATION Added First Added Last 5% 10% 15% 20% Many commercial gradient formers are available. These devices usually load a tube by allowing the gradient solutions to run down the side of the tube. The heaviest concentration is loaded first, followed by successively lighter concentrations. This method is acceptable for wettable tubes; however, loading a nonwettable tube (such as Ultra- Clear, polyallomer, 2 and polycarbonate) by allowing solutions to run down the side of the tube can cause mixing. Gradients in nonwettable tubes can be prepared using a gradient former by placing a long syringe needle or tubing to the tube bottom and reversing the gradient chambers. In that way the lightest gradient concentration is loaded first, underlayed by increasingly heavier concentrations. 1 A complete list of tubes, bottles, and adapters is provided in the latest edition of the Beckman Coulter Ultracentrifuge Rotors, Tubes & Accessories catalog (BR-8101), available at www.beckmancoulter.com. 2 It has been reported, however, that polyallomer tubes have been made wettable by soaking them in a chromic acid bath for about 30 minutes (see Preparation of Polyallomer Centrifuge Tubes for Density Gradients, Anal. Biochem. 32:334-339. H. Wallace, 1969). Also, a method of making Ultra-Clear tubes wettable that has proven successful for some users is described at the end of this section. Tabletop Ultracentrifuge Rotors and Tubes 3-1
Using Tubes and Accessories You can also prepare preformed step gradients by hand, using a pipette. Carefully layer solutions of decreasing concentration by placing the tip of the pipette at the angle formed by the tube wall and the meniscus, or float the lighter gradient concentrations up by adding increased density solutions to the tube bottom using a hypodermic syringe with a long needle such as a pipetting needle. 20 to 22 Gauge Needle 2 to 3 mm 1-mL Syringe 45° to 50° Gradient Another way to form a linear gradient is to allow a step gradient to diffuse to linearity. Depending on the concentration differential between steps and the cross-sectional area, allow 3 to 6 hours for diffusion at room temperature, and about 16 hours at 0 to 4°C. For diffusion of step gradient in Quick-Seal and capped straightwall tubes, slowly lay the tube on its side (tube contents will not spill, but make sure the tube does not roll). After 2 hours at room temperature, slowly set the tube upright. Once the gradient is prepared, layer the sample on top of the gradient. Buffer Gradient 2 to 3 mm Sample with 2 to 3% Sucrose Added For thinwall tubes only partially filled with gradient, add a buffer solution to fill the tube to provide tube wall support. Although the gradient volume is reduced, sample volume is not changed. ➠ NOTE If a partially filled thickwall tube is centrifuged, the tube does not require liquid support, and therefore, the buffer solution is not required. CESIUM CHLORIDE GRADIENTS Cesium chloride gradients can be made by filling the tube with a homogeneous solution of CsCl and sample. Select a homogeneous CsCl solution density so that when it is distributed, its density range will encompass the density of the particle(s) of interest. Refer to Appendix B for an explanation of the use of the CsCl curves. 3-2 Tabletop Ultracentrifuge Rotors and Tubes
Page 1 and 2: TLR-IM-9 ROTORS AND TUBES For Beckm
Page 3 and 4: Mechanical Safety • Use only comp
Page 5 and 6: SCOPE OF THIS MANUAL This manual co
Page 7 and 8: Contents Page Cellulose Propionate
Page 9 and 10: Contents Page Tube and Accessory Ca
Page 11 and 12: Tables Page Table 1-1. Rotors Used
Page 13 and 14: Rotors Fixed Angle Rotor Swinging B
Page 15 and 16: Rotors ! CAUTION In all tabletop ul
Page 17 and 18: Rotors • Near vertical tube rotor
Page 19 and 20: Rotors Table 1-1. Rotors Used in Be
Page 21 and 22: Rotors where s is the sedimentation
Page 23 and 24: Rotors The gradient may be preforme
Page 25 and 26: Rotors ROTOR BALANCE The mass of a
Page 27 and 28: Rotors • Cesium Chloride Gradient
Page 29 and 30: Tubes and Accessories thinwall poly
Page 31 and 32: Tubes and Accessories POLYALLOMER T
Page 33 and 34: Tubes and Accessories organic solve
Page 35: Tubes and Accessories SPACERS AND F
Page 39 and 40: Using Tubes and Accessories Table 3
Page 41 and 42: Using Tubes and Accessories ➠ NOT
Page 43 and 44: Using Tubes and Accessories ! CAUTI
Page 45 and 46: Using Tubes and Accessories METHOD
Page 47 and 48: Using Tubes and Accessories Polycar
Page 49 and 50: Using Tubes and Accessories Spacer
Page 51 and 52: Using Tubes and Accessories QUICK-S
Page 53 and 54: 4 Using Rotors This section contain
Page 55 and 56: Using Rotors ROTOR PREPARATION AND
Page 57 and 58: Using Rotors ! CAUTION In all table
Page 59 and 60: Using Rotors ROTOR PREPARATION AND
Page 61 and 62: Using Rotors OPERATION For runs at
Page 63 and 64: Using Rotors VERTICAL TUBE ROTORS D
Page 65 and 66: Using Rotors Rotor Preparation and
Page 67 and 68: Using Rotors 3. Refer to the centri
Page 69 and 70: Care and Maintenance CLEANING Wash
Page 71 and 72: Care and Maintenance STERILIZATION
Page 73 and 74: Care and Maintenance TUBE AND ACCES
Page 75 and 76: Care and Maintenance ! CAUTION Do n
Page 77 and 78: Care and Maintenance RETURNING A RO
Page 79 and 80: Care and Maintenance SYMPTOM POSSIB
Page 81 and 82: Appendix B The Use of Cesium Chlori
Page 83 and 84: The Use of Cesium Chloride Curves 1
Page 85 and 86: The Use of Cesium Chloride Curves T
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Appendix C Gradient Materials This
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Gradient Materials Table C-2. Densi
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Gradient Materials Table C-4. Densi
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Glossary of Terms clearing factor,
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Glossary of Terms Radel polyphenyls
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Appendix E References Documents ref
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References DS-640 Formation of Line
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ULTRACENTRIFUGE ROTOR WARRANTY All
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