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Rotors and Tubes

1. Ultem is a registered trademark of GE Plastics 3 6 Preparative Ultracentrifuge Rotors and Tubes SEATING THE TUBE PLUGS 1 Cae O ring No fluid appears as wide black line above O ring Preparative Ultracentrifuge Rotors and Tubes Using Tubes Bottles and Accessories 2 After filling the tube make sure that there is no fluid in the stem Draw off excess fluid with a syringe or pipette If necessary wipe the inside of the stem with a lintless tissue 3 Fill the remaining tubes in the same manner Eight tubes can be prepared for use at once in the specially designed racks listed in Table 3 2 gt NOTE The Ultem spacers 361678 snap onto the 3 3 mL tubes 361627 To avoid disturbing the sample or splashing out liquid put the spacers on these tubes before inserting the plugs 1 Make sure that no fluid is in the tube stem and that the stem is clean and dry 2 Insert a Noryl plug assembly plug and O ring shipped assembled in each tube stem 3 Set the plug seating bar on the rack ensuring that the pegs at each end fit into the rack openings 4 Press firmly straight down all along the top of the bar When you remove the bar the plugs should be straight and seated into the stems 5 Check the tubes to be sure all plugs are seated If any plugs are not seated seat them individually 3 7 Using Tubes Bottles and Accessories FILLING AND SEALIN
2. 3 4 Table 3 2 OptiSeal Tubes and Accessories 0 0 0 0 0 e eee eee eee eee 3 6 Table 3 3 Tube Cap Assemblies for Open Top Tubes in Fixed Angle Rotors 3 16 Table 3 4 Required Tools and Torque Values 0 0 00 eee eee 3 21 Table 3 5 Available Bottles Assembly and Operation 3 24 Table 4 1 General Specifications for Beckman Coulter Preparative Fixed Angle Rotots eeren rere ee Re ee 4 3 Table 4 2 Maximum Run Speeds and Tube Volumes for Uncapped Tubes in Fixed Angle Rotors 0 0 0c eee ee 4 5 Table 5 1 General Specifications for Beckman Coulter Preparative Swinging Bucket Rotors 0 0 0 eee 5 3 Table 6 1 General Specifications for Beckman Coulter Preparative Vertical Tube and Near Vertical Tube Rotors 4 6 2 Table 6 2 Rotor Plugs and Tools Used for Vertical Tube and Near Vertical Tube Rotors 0 0 eee eee eee eee 6 7 Preparative Ultracentrifuge Rotors and Tubes Table 7 1 Table D 1 Table D 2 Table D 3 Table D 4 Tube and Bottle Sterilization and Disinfection 7 8 Commonly Used Gradient Materials with Their Solvents D 2 Density Refractive Index and Concentration Data Cesium Chloride at 25 C Molecular Weight 168 37 D 3 Density Refractive Index and Concentration Data Sucrose at 20 C Molecular Weight 342 3 0 0 eee eee eee D 4 Density Conve
3. Caps not properly secured Caps must be properly seated on tubes and then fully tightened Cap components not dry before assembly Thoroughly dry all components before assembling The setscrew may not be sealing the filling hole The nylon insert may have been driven out by the filling hole setscrew Check hex cavity If the threads of the screw are stripped replace the screw It may be necessary to replace the stem also The interface between the setscrew and the nylon insert is critical Refer to insert replacement procedures in this section Insufficient liquid in tube Observe minimum fill volumes Tube too full the meniscus must be kept lower to prevent leakage Tube volume exceeds maximum uncapped volume Refer to the rotor manual for tube volumes and speed reductions Improperly plugged Make sure that no fluid is trapped in the tube stem and that the stem is clean and dry before inserting plug Refer to publication IN 189 for instructions on filling and plugging OptiSeal tubes Improperly sealed After heat sealing squeeze the tube gently if the tube contents may be disturbed to test the seal for leaks If the tube leaks reseal it Tubes may crack or become brittle if they are used below their lower temperature limit Before using tubes at other than stated temperature limits evaluate them under centrifugation conditions If sample is frozen in tubes make sure that tubes are thawed to at least 2 C before ce
4. Thickwall Tubes Fill at least half full Fixed Angle Rotors Fill all opposing tubes to the same level Thinwall Tubes Must be completely filled liquid and cap for support of the tube wall is critical e Thickwall Tubes Can be partially filled and centrifuged as indicated in the applicable rotor manual Speed reductions may be required for these partially filled tubes For greater fill volumes and faster speeds tube caps should be used Refer to the applicable rotor manual for fill volumes and speed limitations OTHER OPEN TOP TUBES Open top tubes of other materials can also be used in fixed angle and swinging bucket rotors Vertical tube and near vertical tube rotors use only OptiSeal or Quick Seal tubes Fill these tubes as indicated below 3 12 Preparative Ultracentrifuge Rotors and Tubes Using Tubes Bottles and Accessories Polycarbonate Thickwall polycarbonate tubes can be centrifuged partially filled Observe maximum rotor speeds and fill volumes listed in the applicable rotor manual UltraClear Fill all opposing tubes to the same level For swinging bucket rotors fill to within 2 or 3 mm of the top of the tube Hil thickwall polypropylene tubes at least half full to maximum level in fixed angle rotors Speed reduction is required Refer to the applicable rotor manual Polypropylene Fill all opposing tubes to the same level For swinging bucket rotors fill to within 2 or 3 mm o
5. Je Tube Cap Crown Inspect Here Setscrew Insert ASSEMBLING TUBE CAPS Preparative Ultracentrifuge Rotors and Tubes 1 Inspect cap components before each use Replace any damaged components Inspect the cap crown for stress cracking and check the stem and nut threads for damage or signs of wear and for adequate lubrication Inspect the O ring or gasket for cracks nicks or flattened areas Inspect the underside of the stem the white nylon insert should not protrude below the filling hole If the cap assembly has a filling hole run the setscrew in against the nylon insert making sure the setscrew will not displace the insert Check the setscrew hex socket for damage that would prevent tightening or removal Regularly apply a thin uniform coat of Spinkote lubricant 306812 on the stem threads III NOTE Keep the O ring or flat gasket dry and free from lubricant during assembly Wet or greased O rings or gaskets will slip when the cap nut is tightened and the cap will not seal properly See Figure 3 3 and Table 3 4 for required tools and torque requirements N CAUTION Do not use damaged wrenches or hex drivers or tools that have burrs A burred tool can score the crown which could then fail and damage the rotor Torque Wrench Nylon Insert d Tool 302460 Hex Driver ve 841883 Hex Driver ue 841884 es Socket 858123 S Socket 870432 x Socke
6. 355601 357001 50 40 Type 16 14 000 356977 Type 28 20 000 b 29x 102 polypropylene 355601 3570004 50 40 Type 16 14 000 356977 Type 16 14 000 356977 b 29 x 102 3570034 50 40 Type 28 20 000 aluminum 355623 355622 70 35a Type 45 Ti 45 000 355655 38 x 102 Type 35 35 000 Noryl 355621 355620 70 35 Type 21 21 000 b 52 x 134 355674 150 150 Type 16 5 000 339362 355627 60 x 120 Delrin 362247 334025 250 250 Type 19 19 000 w Noryl plug 358275 64 x 124 Noryl 358977 356013 250 190 Type 16 16 000 b 32 x 124 355666 250 190 Type 16 14 000 334915 3583264 64x 124 Noryl 358977 356011 250 190 Type 16 16 000 a Several rotors must be centrifuged at reduced speeds when bottles are filled below maximum fill volume Types 90 Ti 80 Ti 75 Ti 70 1 Ti and 65 at 60 000 rpm Types 70 Ti 60 Ti 55 2 Ti and 50 2 Ti at 50 000 rpm Type 45 Ti at 35 000 rpm Available only as bottle and cap assembly Above 20 000 rpm insert assembly 355601 must be used 4 Polyallomer e Polypropyle ne Preparative Ultracentrifuge Rotors and Tubes SAMPLE RECOVERY CAPPED TUBES OptiSeal TUBES Preparative Ultracentrifuge Rotors and Tubes Using Tubes Bottles and Accessories N CAUTION If disassembly reveals evidence of leakage you should assume that some fluid escaped the container or rotor Apply appropriate decontam ination procedures to the centrifuge rotor
7. Model L7 s and Optima L s all classified R Optima XL s and L XP s classified S ROTORS THAT MAY BE USED IN THIS INSTRUMENT Type 50 Ti Type 50 2 Ti Type 50 3 Ti Type 50 Type 45 Ti Type 40 Type 40 2 Type 40 3 Type 25 Type 15 SW 50 1 SW 41 Ti SW 28 SW 28 1 SW 27 SW 27 1 SW 25 1 SW 25 2 and zonals Type 50 Ti Type 50 2 Ti Type 50 3 Ti Type 50 Type 45 Ti Type 40 Type 40 2 Type 40 3 Type 25 SW 50 1 SW 41 Ti SW 40 Ti SW 30 SW 30 1 SW 28 SW 28 1 SW 27 SW 27 1 SW 25 1 SW 25 2 and zonals Type 50 Ti Type 50 2 Ti Type 50 3 Ti Type 50 Type 45 Ti Type 40 Type 40 2 Type 40 3 Type 25 SW 50 1 SW 41 Ti SW 30 SW 30 1 SW 28 SW 28 1 SW 27 SW 27 1 SW 25 1 SW 25 2 and zonals Type 50 Ti Type 50 2 Ti Type 50 3 Ti Type 50 Type 45 Ti Type 42 2 Ti Type 40 Type 40 2 Type 40 3 Type 25 SW 50 1 SW 41 Ti SW 30 SW 30 1 SW 28 SW 28 1 SW 27 SW 27 1 SW 25 1 SW 25 2 and zonals Type 50 Ti Type 50 3 Ti Type 50 Type 45 Ti Type 40 Type 40 2 Type 40 3 SW 50 1 SW 30 SW 30 1 SW 25 1 and zonals Any Beckman Coulter preparative rotor including zonal and continuous flow rotors EXCEPT the following a all Type 15 rotors and b all Type 35 and Type 42 1 rotors with serial numbers 1299 or lower see Special Action below Type 16 and Type 2
8. Type 50 Ti Type 50 3 Ti Type 50 Type 40 Type 40 2 Type 40 3 Type 25 Type 15 SW 41 Ti SW 30 SW 30 1 SW 28 SW 28 1 SW 27 SW 27 1 SW 25 1 SW 25 2 and zonals All Model L2 50 s classified D Type 50 Ti Type 50 3 Ti Type 50 Type 40 Type 40 2 Type 40 3 Type 25 Type 15 SW 50 1 SW 41 Ti SW 30 SW 30 1 SW 28 SW 28 1 SW 27 SW 27 1 SW 25 1 SW 25 2 and zonals All Model L2 50 s classified F Type 50 Ti Type 50 2 Ti Type 50 3 Ti Type 50 4 Ti Type 50 Type 45 Ti Type 40 Type 40 2 Type 40 3 Type 25 Type 15 SW 50 1 SW 41 Ti SW 30 SW 30 1 SW 28 SW 28 1 SW 27 SW 27 1 SW 25 1 SW 25 2 and zonals All Model L2 65 s classified D Type 50 Ti Type 50 3 Ti Type 50 Type 40 Type 40 2 Type 40 3 Type 25 Type 15 SW 50 1 SW 41 Ti SW 30 SW 30 1 SW 28 SW 28 1 SW 27 SW 27 1 SW 25 1 SW 25 2 and zonals To the maximum speed of the ultracentrifuge as applicable Preparative Ultracentrifuge Rotors and Tubes xii Classification Program xiii INSTRUMENT CLASSIFICATION All Model L2 65 s classified F All Model L2 65B s and Model L2 75B s classified G All Model L3 40 s and Model L3 50 5 classified F All Model L3 40 s and Model L3 50 s classified G Model L4 s classified Q Model L5 s L5B s L8 s and L8M s all classified H
9. Using Tubes Bottles and Accessories c Apply the tip of the Tube Topper vertically to the seal former Press down gently for about 10 seconds The seal guide should move down the tube stem until it rests on the tube shoulder Using the seal guide prevents the seal former from being pressed into the tube shoulder III NOTE Always apply the tip of the Tube Topper verti cally to the seal former Apply gentle pressure when sealing the tube d When the seal guide has moved to the correct position remove the Tube Topper and pinch the circular seal guide to hold the seal former in place e Place the heat sink small end over the cap for 2 to 3 seconds while the plastic cools do NOT let the seal former pop up If the seal former does pop up the tube may not have an adequate seal and may need to be resealed f Remove the heat sink and seal guide When the seal former cools remove it by hand or with the removal tool 361668 Save the seal guide and former for future use 3 10 Preparative Ultracentrifuge Rotors and Tubes Using Tubes Bottles and Accessories METHOD B WITHOUT THE SEAL GUIDE gt NOTE Always apply the tip of the Tube Topper verti cally to the seal former Apply gentle pressure when sealing the tube a Turn the Tube Topper pushbutton to USE position Press the pushbutton and wait 3 to 5 seconds for the tip to heat b Apply the tip of the Tube Topper vertically to the seal
10. 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 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 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 gt 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 3 2 Cesium chloride gradients can be made by filling the tube with a homogeneous solution of CsCl and sample Select a homogeneous CsCI solution density so that when it is di
11. Fax 31 297 288082 SINGAPORE Beckman Coulter Singapore Pte Ltd 116 Changi Road Unit 03 01 02 Singapore 419718 Telephone 65 6339 3633 Fax 65 6336 6303 SOUTH AFRICA SUB SAHARAN AFRICA Beckman Coulter Stand 1A Primegro Park Tonetti Street 1685 Halfway House Johannesburg Republic of South Africa Telephone 27 11 805 2014 5 Fax 27 11 805 4120 e mail beckman intekom co za SPAIN Beckman Coulter Espafia S A C Caleruega 81 28033 Madrid Spain Telephone 34 91 3836080 Fax 34 91 3836096 email bioresearch espana beckman com SWEDEN Beckman Coulter AB Archimedesvaegen 7 Box 111 56 SE 168 11 Bromma Sweden Telephone 46 8 564 85 900 Telefax 46 8 564 85 901 SWITZERLAND Beckman Coulter International S A 22 Rue Juste Olivier Case Postale 301 303 CH 1260 Nyon Switzerland Telephone 41 0800 850 810 Fax 41 0848 850 810 TAIWAN Beckman Coulter Taiwan Inc Taiwan Branch 8th Floor 216 Tun Hwa South Road Section 2 Taipei 106 Taiwan Republic of China Telephone 886 2 2378 3456 Fax 886 2 2377 0408 TURKEY Beckman Coulter Ltd E 5 Yanyol Faith Cad 81410 Soganlik Kartal Istanbul Turkey Telephone 90 216 309 1900 Fax 90 216 309 0090 UNITED KINGDOM Beckman Coulter United Kingdom Ltd Oakley Court Kingsmead Business Park London Road High Wycombe Buckinghamshire HP11 1JU England U K Telephone 44 01494 441181 Fax 44 01494 44
12. and syringe Slice the tube using the Beckman CentriTube Slicer 347960 Refer to publication L TB 010 for instructions for using the CentriTube Slicer For additional information on fraction recovery systems available from Beckman Coulter refer to the latest edition of Ultracentrifuge Rotors Tubes amp Accessories publication BR 8101 MAKING ULTRA CLEAR TUBES WETTABLE Preparative Ultracentrifuge Rotors and Tubes The following method of making Ultra Clear tubes wettable has proven successful for some users 1 Polyvinyl alcohol 2 g was dissolved in distilled water 50 mL by stirring and heating to gentle reflux 2 Isopropanol 50 mL was slowly added to the hot solution and stirring and heating continued until a clear solution was obtained 3 The solution was then allowed to cool to room temperature 3 29 Using Tubes Bottles and Accessories 4 Ultra Clear tubes were filled with the coating solution then aspirated out with a water pump after 15 minutes leaving a thin film on the tube walls A small amount of solution that collected in the tube bottoms after standing was removed with a pipette 5 The tubes were left open to dry at room temperature overnight then filled with distilled water After standing overnight at room temperature the distilled water was poured out 6 Finally the tubes were briefly flushed with water tapped to remove excess liquid and left to dry 3 30 Preparative Ultracentrif
13. gt NOTE In swinging bucket rotors the interband separa tion after centrifugation is the same as during centrifugation as there is no gradient reorienta tion In fixed angle near vertical tube and vertical tube rotors the gradient must reorient to a horizontal position after centrifugation Therefore to determine the interband separation after centrifugation when the tube is held upright dup use d do C l UP cosQ cD where d is the interband separation achieved during centrifugation and 9 is the tube angle Example B Knowing particle densities 1 50 and 1 52 g mL how do you achieve good separation In Figure C 2 sketch in a horizontal line corresponding to each particle s buoyant density Select the curve at the desired temperature 4 C and tube volume full that gives good separation Note the speed indicated along the curve 50 000 rpm The Use of Cesium Chloride Curves C 6 4 From Figure C 1 determine the maximum allowable homoge neous CsCl density that corresponds to the selected temperature speed and fill volume from Figure C 2 in this case 1 51 g mL In this example particles will band at about 56 and 58 mm from the axis of rotation about 2 mm of interband separation at the tube angle When the tube is held upright there will be about 2 21 mm of interband separation To determine the interband volume in millimeters use V 2nr h C 2 where r is the tube radiu
14. 2 Contents Page ROTORS General Description sse accedere sariseu Eger e Rr a 1 1 Rotor Designations lssleleeeeee e 1 1 Materials scenes eee ect ere pr e REPE oe 1 3 Drive PMS 222b reel ergo eb eei OR p 1 3 Rotor Selection lseeeleeeeee eh 1 4 Pelleting Differential Separation 0 0 1 8 Isopycnic Separations 0 0 0 eee eee eee eee 1 11 Rate Zonal Separations 0 0 cee eee ee eee 1 12 General Operating Information 000002 e eee 1 13 Rotor Balance 1 0 ene ene 1 13 Overspeed Protection 0 0 0 cece eee eee 1 14 Allowable Run Speeds 00 0 0 c eee eee eee eee 1 16 TUBES BOTTLES AND ACCESSORIES Labware Selection Criteria 2 0 0 eee eee eee ee 2 1 Labware Material Compatibility with Solvents and Sample 2 3 Gradient Formation and Fractionation 2 3 Labware Lypes 4i cane dee Ree xd ewe ee bee 2 3 Polyallomer Tubes 0 0 0 cece eee e 2 4 Polycarbonate Tubes 0 0 0 c eee eee eee 2 5 Polypropylene Tubes 0 0 0 cece ee ee eee 2 5 Polyethylene Tubes 0 0 0 0 cece ee eee eee ee 2 6 Ultra Clear Tubes 000 cece ieee eee e 2 6 Preparative Ultracentrifuge Rotors and Tubes iii Contents SECTION 3 Page Cellulose Propionate Tubes 0 0 0 c eee eee 2 6 Stainless Steel Tubes 2 0 0 0 cee eee eee 2 7 konical TUDES 5rd dh be do aora 2 7 B
15. Bovine Serum Albumin Pelleting Using the ESP Pelleting Simulation from the Optima eXPert Software A 1941 Predicting Protein Separation in Rate Zonal Centrifugation Using the E Run simulation from the Optima eXPert Software A 2038 Preparation of Intestinal Mucins Using the NVT 65 Near Vertical Tube Rotor AR 8093 Fast Separations of Plasmid DNA Using Discontinuous Gradients in the Preparative Ultracentrifuge BA99 60495 Rotor Safety Guide Warranty and Care BR 8101 Ultracentrifuge Rotors Tubes amp Accessories Catalog BR 9272 Optima L XP DS 468 Techniques of Preparative Zonal and Continuous Flow Ultracentrifugation DS 514 Ultracentrifuge Methods for Lipoprotein Research DS 528 Use of the w t Integrator DS 694 30 Minute 2 Step Purification of Plasma Membranes from Cultured Cells DS 709 g Max System Short Pathlengths in High Force Fields DS 728 Optimizing Centrifugal Separations Sample Loading Preparative Ultracentrifuge Rotors and Tubes Preparative Ultracentrifuge Rotors and Tubes DS 770 DS 793 DS 887 DS 9271 DS 9338 DS 9339 DS 9340 DS 9343 SR 171 SR 182 References Plasmid Separations in NVT Near Vertical Tube Rotors Preparation of Intestinal Mucins Using the NVT 65 Near Vertical Tube Rotor Obtain Greater Purity and Efficiency with the New NVT 65 2 Rotor Using k Factor to Compare Rotor Efficiency Optima L 80 XP Ultracentrifuge Datasheet Optima L 90K Ultracent
16. Maintenance I gt NOTE Do not soak aluminum cap parts in a strong detergent solution as the anodizing may be attacked 2 Clean the nut and stem threads regularly with concentrated Solution 555 and a brush 3 Rinse all parts in distilled water and dry them 4 Apply a thin even coat of Spinkote lubricant 306812 to the stem threads 5 Wipe O rings and gaskets clean with a tissue Do not lubricate O rings or gaskets DECONTAMINATION e If the tube caps become contaminated with radioactive material decontaminate them using a solution that will not damage the anodized surfaces Beckman Coulter has tested a number of solutions and found two that do not harm anodized aluminum RadCon Surface Spray or IsoClean Solution for soaking and Radiacwash gt NOTE IsoClean can cause fading of colored anodized surfaces Use it only when necessary and remove it promptly from surfaces While Beckman Coulter has tested these methods and found that they do not damage components no guarantee of sterility or disin fection is expressed or implied Consult your laboratory safety officer regarding the proper decontamination methods to use n U S contact Nuclear Associates New York in Eastern Europe and Commonwealth States contact Victoreen GmbH Munich in South Pacific contact Gammasonics Pty Ltd Australia in Japan contact Toyo Medic Co Ltd Tokyo 5 In U S contact Biodex Medical Systems Shirley New York in
17. O P Q and some F with the mechanical overspeed system have a knockout pin in the rotor chamber Rotors that are equipped for the mechanical system have overspeed cartridges installed in the sides of the rotor base If overspeeding occurs a small pin is forced out of the cartridge and knocks out the overspeed pin in the chamber causing the instrument to shut down N CAUTION Rotors without mechanical overspeed cartridges should not be used in ultracentrifuges classified other than G H R or S The overspeed device should be replaced if a rotor is regularly being used at speeds below its rated speed due to the use of adapters stainless steel tubes CsCl gradients etc Instructions for replacing overspeed disks are provided in Section 7 of this manual Rotors ALLOWABLE RUN SPEEDS Under some conditions the maximum allowable speed of the rotor indicated by the rotor name must be reduced to ensure that neither the rotor nor the labware are overstressed during centrifugation Check the recommended run speed for your rotor before centrifuging dense solutions CsCl gradients stainless steel tubes polycarbonate bottles uncapped plastic tubes in fixed angle rotors and sleeve type adapters Dense Solutions To protect the rotor from excessive stresses due to the added load reduce run speed when centrifuging a solution with a density greater than the allowable density rating of the rotor specified in the rotor instruction ma
18. Polyallomer mL Polycarbonate Polyallomer Rotor Type 7x20 343775 343621 230 uL 42 000 42 000 42 2 Ti 7x20 342303 230 uL 42 000 42 2 Ti 45 000 50 4 Ti 8x 51 355657 1 25 000 25 11x 89 355632 355641 3 5 30 000 30 000 45 Ti 35 13 x 64 355645 355644 4 50 000 30 000 50 4 Ti 50 3 Ti 40 3 6 5 50 000 50 000 50 50 000 30 000 80 Ti 75 Ti 16 x 64 355647 355646 70 1 Ti 50 Ti 40 000 30 000 40 7 5 50 000 30 000 65 50 Ti 16 x 76 355630 355640 55 000 29 000 Sa 16 5 45 000 20 000 70 Ti 60 Ti 55 2 Ti 50 2 Ti 24 40 000 20 000 42 1 25 x 89 355631 355642 18 30 000 20 000 30 25 x 102 335432 12 6 000 28 28 x 102 357006 357007 35 20 000 20 000 28 44 30 000 15 000 45 Ti 35 38 x 102 355628 355643 34 21 000 15 000 21 Maximum speeds are those for capless tubes tested at 25 C for 24 hours Rotors are not listed for tubes used with adapters Cellulose propionate Preparative Ultracentrifuge Rotors and Tubes 4 5 Using Fixed Angle Rotors PRERUN SAFETY CHECKS N 20 Sector 6 355539 m n Read all safety information in the rotor manual before using the rotor 1 Make sure that the rotor and lid are clean and show no signs of corrosion or cracking Make sure the rotor is equipped with the correct overspeed disk refer to Section 1 If the disk is missing or damaged replace it as described in Section 7 Check the chemical compatibilities of all materials
19. Tubes Using Tubes Bottles and Accessories AN CAUTION Before plugging in the Tube Topper be sure that you have a proper power source 120 V 50 or 60 Hz Charge your Cordless Tube Topper only in the charging stand supplied with it 1 Remove the Tube Topper from the charging stand Leave the pushbutton turned to LOCK position N WARNING Touching the heated tip of the Tube Topper will cause burns When the pushbutton is pressed the tip heats almost immediately Make sure the pushbutton is turned to LOCK position unless you are actually sealing a tube Seal Former 2 Place a seal former on each tube stem The Teflon coating on the seal formers is permanent Do not scratch the interior of the formers as you may damage this coating 3 Seal each tube using Method A or B Method A is preferable when sealing smaller tubes or when resealing a tube that leaks A CAUTION Always keep the Tube Topper in its charging stand when not in use Do not lay the unit against any surface after use until the tip has cooled 3 to 5 minutes after shut off METHOD A WITH THE SEAL GUIDE Seal Guid j i j a Place a seal guide with the flat side down over the seal 3 former i DQ b Turn the Tube Topper pushbutton to USE position Press the pushbutton and wait 3 to 5 seconds for the tip to heat 4 Teflon is a registered trademark of E I Du Pont de Nemours amp Co Preparative Ultracentrifuge Rotors and Tubes 3 9
20. a tube that has become yellowed or brittle with age or Grazing excess exposure to ultraviolet light e Crazing the appearance of fine cracks on tubes and bottles is the result of stress relaxation If a crack approaches the outer wall Cracking of the tube or bottle discard it e Discard any deformed or cracked adapters TUBE AND BOTTLE STORAGE Tubes and bottles have an indefinite shelf life if properly stored Store in a dark cool dry place away from ozone chemical fumes and ultraviolet light sources REMOVING JAMMED OR COLLAPSED TUBES Centrifugal force may collapse improperly sealed or capped thinwall tubes Observe careful filling and capping procedures to prevent tube collapse I gt NOTE Centrifugation often causes a slight vacuum to build up in the tube cavity occasionally resulting in a suction effect when removing the tubes from the rotor This effect is especially pronounced in a rotor that has been centrifuged at low temperature A brief delay approxi mately 5 minutes after the rotor comes to rest before removing the tubes can make tube removal easier If tubes are difficult to remove from the rotor use a gentle twisting or rocking motion and remove the tube slowly to avoid sample mixing Preparative Ultracentrifuge Rotors and Tubes 7 9 Care and Maintenance TUBE CAP CARE CLEANING If a tube is jammed or collapsed in the rotor try one of the following techniques but DO NOT force the tu
21. atmospheric pressure inside the rotor during centrifugation if they are properly lubricated Some rotors have fluted bodies designed to eliminate unnecessary weight and minimize stresses 4 1 Using Fixed Angle Rotors Axis of Rotation Axis of Rotation Axis of Rotation fee Tmin fav max z 1st Row 2nd Row 3rd Row Axis of Rotation 4 2 Type 100 Ti Type 70 1 Ti Type 50 4 Ti Type 25 Figure 4 1 Fixed Angle Rotors Preparative Ultracentrifuge Rotors and Tubes Using Fixed Angle Rotors Table 4 1 General Specifications for Beckman Coulter Preparative Fixed Angle Rotors Rotors in parentheses are no longer manufactured Relative Radial Distances mm Number of Maximum Centrifugal Tube Tubes x Tube Rotor Speed Field x g Angle k Capacity Type rpm at less degrees Tmax Tay Tin Factor mL 100 Ti 100 000 802 400 26 71 6 55 5 39 5 15 8 x 6 8 90 Ti 90 000 694 000 25 76 5 55 4 34 2 25 8x 13 5 80 Ti 80 000 602 000 25 5 84 0 62 5 41 0 28 8 x 13 5 75 Ti 75 000 502 000 25 5 79 7 58 3 36 9 35 8 x 13 5 70 1 Ti 70 000 450 000 24 82 0 61 2 40 5 36 12 x 13 5 70 Ti 70 000 504 000 23 91 9 65 7 39 5 44 8 x 38 5 65 65 000 368 000 23 5 77 8 57 3 36 8 45 8 x 13 5 60 Ti 60 000 362 000 23 5 89 9 63 4 36 9 63 8 x 38 5 55 2 Ti 55 000 340 000 24 100 3 73 5 4
22. caps are not available for thickwall tubes used in Types 80 Ti 75 Ti 70 1 Ti 50 4 Ti 50 3 Ti 42 2 Ti 40 3 and 25 rotors Tube Cap Assembly Hex Nut Crown Setscrew Insert O ring or Gasket Stem Tube Type Rotor Type 8 mm 5 16 in 303624 303658 303379 303379 303809 303810 303730 303730 303377 303377 UC UC 90 Ti 80 Ti 75 Ti 70 1 Ti 65 50 Ti 50 40 50 3 Ti 13 mm 1 2 in 303113 305022 346256 301870 301870 301870 307004 307004 307004 803543 302312 344672 344672 344672 307005 302331 346246 SS SS thinwall PA UC SS 80 Ti 75 Ti 70 2 Ti 70 Ti 60 Ti 55 2 Ti 50 4 Ti 50 3 Ti 50 2 Ti 50 Ti 45 Ti 42 1 40 35 21 80 Ti 75 Ti 70 1 Ti 70 Ti 65 60 Ti 55 2 Ti 50 4 Ti 50 3 Ti 50 2 Ti 50 Ti 45 Ti 42 1 40 35 21 90 Ti 80 Ti 75 Ti 70 1 Ti 70 Ti 65 60 Ti 55 2 Ti 50 4 Ti 50 3 Ti 50 2 Ti 50 Ti 45 Ti 42 1 40 35 21 16 mm 5 8 in 303319 330860 3389074 341968 301870 301870 301870 335320 307006 330774 338911 335319 338864 803543 338864 338864 302312 302312 302312 302312 301869 858046 878572 858046 870380 302266 330788 338910 341969 341969 SS thinwall PA UC thickwall PA PC thi
23. due to a failure of a Beckman Coulter tube bottle tube cap spacer or adapter covered under the Conditions of this Warranty Beckman Coulter will repair or replace the rotor and repair the instrument as per the conditions in policy point 2 above and the replacement policy 5 Damage to a Beckman Coulter rotor or instrument due to the failure or malfunction of a non Beckman Coulter tube bottle tube cap spacer or adapter is not covered under this warranty although Beckman Coulter will assist in seeking compensation under the manufacturer s warranty Disclaimer IT IS EXPRESSLY AGREED THAT THE ABOVE WAR RANTY SHALL BE IN LIEU OF ALL WARRANTIES OF FIT NESS AND OF THE WARRANTY OF MERCHANTABILITY AND BECKMAN COULTER SHALL HAVE NO LIABILITY FOR SPECIAL OR CONSEQUENTIAL DAMAGES OF ANY KIND WHATSOEVER ARISING OUT OF THE MANUFAC TURE USE SALE HANDLING REPAIR MAINTENANCE OR REPLACEMENT OF THE PRODUCT Factory Rotor Inspection Service Beckman Coulter Inc will provide free mechanical and metallurgical inspection in Palo Alto California USA of any Beckman Coulter rotor at the request of the user Shipping charges to Beckman Coulter are the responsibility of the user Rotors will be inspected in the user s laboratory if the centrifuge in which they are used is covered by an appropriate Beckman Coulter Service Agreement Contact your local Beckman Coulter office for details of service coverage or cost Before shipping cont
24. filling hole setscrew and threads If the hex cavity in the setscrew shows signs of wear replace the setscrew NYLON INSERT REPLACEMENT The nylon insert fits into the cap stem and can become worn or loose with continued cap use If the insert is worn the setscrew will no longer seal the cap and it should be replaced 1 Remove the cap setscrew and fit the nylon insert tool 302460 firmly into the stem 2 Unscrew the insert 3 Fit a new insert 302312 on the end of the tool and screw it into the stem until it bottoms firmly against the stem threads Preparative Ultracentrifuge Rotors and Tubes 7 13 Care and Maintenance RETURNING A ROTOR OR ACCESSORY TO THE FACTORY Before returning a rotor or accessory for any reason prior permission a Returned Goods Authorization form must be obtained from Beckman Coulter Inc This RGA form may be obtained from your local Sales Office It should contain the following information serial number history of use approximate frequency of use reason for the return original purchase order number billing number and shipping number if possible name and phone number of the person to be notified upon receipt of the rotor or accessory at the factory and name and phone number of the person to be notified about repair costs etc To protect our personnel it is the customer s responsibilit
25. held in the tube cap vise Clear and black anodized e Clear anodized crown aluminum caps that use O rings for sealing are used in many rotors with many types of tubes Refer to Table 3 3 The caps should be hand tightened with a hex driver while the tube is held in the tube cap vise refer to ASSEMBLING TUBE CAPS below Aluminum caps that use flat gaskets for sealing are used with small diameter 13 mm thinwall Ultra Clear and polyallomer tubes in all fixed angle rotors except Types 42 2 25 and 19 They are also used with stainless steel tubes Some caps for very small diameter less than 13 mm tubes do not have filling holes nylon insert or setscrew The tube crown is made from a lighter weight aluminum alloy than that used for other clear aluminum caps therefore do not interchange cap parts or use these caps in place of O ring caps since the weight difference can cause rotor imbal ance The caps should be hand tightened with a hex driver while the tube is held in the tube cap vise Caps for thickwall tubes used in Type 21 rotors have Delrin crown washers and must be tightened with a torque wrench Caps for thickwall tubes used in Type 30 rotors have black anodized crowns and use neoprene O rings for sealing These caps have Delrin crown washers and must be tightened with a torque wrench 3 18 Preparative Ultracentrifuge Rotors and Tubes Using Tubes Bottles and Accessories INSPECTING AND LUBRICATING TUBE CAPS
26. if they appear to be the same Tube caps are designed specifically for a partic ular tube in a particular rotor Cap stems and crowns are often machined differently for each type of rotor to ensure proper sealing and support and to withstand stresses experienced during centrifugation The uneven weight differ ence between an O ring cap and a comparable flat gasket cap as much as 0 7 gram could damage the rotor Store tube caps assembled dry and classified according to the tube and rotor for which they are designed High strength titanium cap assemblies for thinwall Ultra Clear and polyallomer tubes are required for maximum rotor speeds in the Type 90 Ti 80 Ti 75 Ti and 70 1 Ti rotors Titanium caps can be identified by the darker gray shiny metal The cap crown is specially machined to lock onto the cap stem To ensure proper compression of the O ring these caps must be tightened with a torque wrench while the capped tube is held in the tube cap vise A special crimp lock cap assembly is required to provide the reliable seal necessary for maximum rotor speed in the Type 70 1 Ti rotor The 25 x 83 mm thinwall polyallomer tube is crimped between the titanium crown and the aluminum stem Instructions for assembling the tube and cap are in the Type 70 Ti rotor instruction manual A special tool kit 338841 is required Using Tubes Bottles and Accessories Table 3 3 Tube Cap Assemblies for Open Top Tubes in Fixed Angle Rotors Tube
27. in Section 2 Consult individual rotor manuals for allowable run speeds Preparative Ultracentrifuge Rotors and Tubes Rotors Stainless Steel Tubes Reduce run speed when centrifuging stain less steel tubes to prevent the rotor from overstressing due to the added weight The criteria for speed reduction percentage depends on the tube cap material and the strength of the rotor in use consult the individual rotor manual or publication L5 TB 072 Preparative Ultracentrifuge Rotors and Tubes 1 17 2 Tubes Bottles and Accessories This section describes various labware used in Beckman Coulter preparative rotors General instructions for using containers follow in Section 3 Care and maintenance instructions are in Section 7 General rotor use instructions are in Sections 4 through 6 The indi vidual rotor manual that comes with each rotor provides specific instructions on the tubes bottles and accessories that can be used in a particular rotor A table of chemical resistances can be found in Appendix A of this manual LABWARE SELECTION CRITERIA No single tube or bottle design or material meets all application requirements Labware choice is usually based on a number of factors The centrifugation technique to be used including the rotor in use volume of sample to be centrifuged need for sterilization importance of band visibility and so forth Chemical resistance the nature of the sample and any solvent or grad
28. seals and lubrication are excluded from this warranty and should be frequently inspected and replaced if they become worn or damaged 8 Keeping a rotor log is not mandatory but may be desirable for maintenance of good laboratory practices Preparative Ultracentrifuge Rotors and Tubes Repair and Replacement Policies 1 If a Beckman Coulter rotor is determined by Beckman Coulter to be defective Beckman Coulter will repair or replace it subject to the Warranty Conditions A replacement rotor will be warranted for the time remaining on the original rotor s warranty 2 If a Beckman Coulter centrifuge is damaged due to a failure of a rotor covered by this warranty Beckman Coulter will supply free of charge 1 all centrifuge parts required for repair except the drive unit which will be replaced at the then current price less a credit determined by the total number of revolutions or years completed provided that such a unit was manufactured or rebuilt by Beckman Coulter and ii if the centrifuge is cur rently covered by a Beckman Coulter warranty or Full Service Agreement all labor necessary for repair of the centrifuge 3 If a Beckman Coulter rotor covered by this warranty is dam aged due to a malfunction of a Beckman Coulter ultracentrifuge covered by an Ultracentrifuge System Service Agreement Beckman Coulter will repair or replace the rotor free of charge 4 If a Beckman Coulter rotor covered by this warranty is damaged
29. support for the hydrostatic forces generated by centrifugation Tubes in near vertical tube rotors designated NV are also held at an angle to the axis of rotation in numbered tube cavities However the reduced tube angle of these rotors typically 7 to 10 degrees reduces run times from fixed angle rotors with tube angles of 20 to 45 degrees while allowing components that do not band under sepa ration conditions to either pellet to the bottom or float to the top of the tube As in vertical tube rotors rotor plugs are used in these rotors to restrain the tubes in the cavities and provide support for the hydro static forces generated by centrifugation 1 2 Preparative Ultracentrifuge Rotors and Tubes MATERIALS DRIVE PINS THU Adapter 7Z Drive Pin Preparative Ultracentrifuge Rotors and Tubes Rotors Beckman Coulter rotors are made from either aluminum or titanium or from fiber reinforced composites A titanium rotor is designated by T or Ti as in the Type 100 Ti the SW 55 Ti or the NVT 90 rotor A fiber composite rotor is designated by C as in VC 53 and an aluminum composite rotor is designated by AC as in VAC 50 Rotors without the T Ti C or AC designation such as the Type 25 are fabricated from an aluminum alloy Titanium rotors are stronger and more chemical resistant than the aluminum rotors Exterior surfaces of titanium and composite rotors are finishe
30. the straightedge intersects the third column Rotors and polycarbonate tubes The k factors for half filled tubes can be calculated by using an approximate Fmax and r4 in k factor equation 1 ISOPYCNIC SEPARATIONS A sedimentation equilibrium or isopycnic method separates parti cles on the basis of particle buoyant density Each component in the sample travels through the gradient until it reaches an equilibrium position Particle velocity due to differences in density is given in the following expression d p Po E DUO NNUS v l 18u E g 6 where v sedimentation velocity dr dt d particle diameter Pp particle density Pe solution density u viscosity of liquid media g standard acceleration of gravity At equilibrium p p is zero and particle velocity is therefore zero The gradient may be preformed before the run or generated during centrifugation For gradients formed by centrifugation the time it takes to form a gradient depends on the sedimentation and diffusion coefficients of the gradient material the pathlength and the rotor speed For a given gradient material the shorter the pathlength and the higher the rotor speed the faster the gradient will form In general the time required for gradients to reach equilibrium in swinging bucket rotors will be longer than in fixed angle rotors One way to reduce run times is to use partially filled tubes Refer to the appropriate rotor instruction man
31. three piece cap assembly consisting of a Noryl plug a neoprene O ring and a Delrin cap Information about these bottles can be found in the individual rotor manuals Preparative Ultracentrifuge Rotors and Tubes 2 7 Tubes Bottles and Accessories TEMPERATURE LIMITS Each labware material has a specified temperature range Although some high speed centrifuges can achieve temperatures as high as 45 C only certain tube or bottle materials can be run under these conditions Most containers are made of thermoplastic materials that soften at elevated temperatures This temperature induced softening together with such factors as the centrifugal force the run duration the type of rotor previous run history and the tube angle can cause labware to collapse Therefore if high temperature runs above 25 C are required it is best to pretest labware under the actual experimental conditions using buffer or gradient of similar density rather than a valuable sample Stainless steel tubes can be centri fuged at any temperature 25 C Qmm gt Plastic labware has been centrifuge tested for use at temperatures between 2 and 25 C For centrifugation at other temperatures pretest tubes under anticipated run conditions If plastic containers are frozen before use make sure that they are thawed to at least 2 C prior to centrifugation SPACERS AND FLOATING SPACERS e OptiSeal tubes must be used with the appropriate spacer
32. to seal properly OptiSeal spacers are listed in Table 3 2 Spacer e Quick Seal tubes use a spacer Table 2 2 one or more floating pond spacers or a combination of both depending on the size of the tube to support the top of the tube during centrifugation The particular combination depends on the type of rotor being used In swinging bucket and fixed angle rotors the top of the tube must be supported In near vertical tube and vertical tube rotors the entire tube cavity must be filled The g Max system uses a combination of short bell top Quick Seal tubes and floating spacers also referred to as g Max spacers The floating spacers sit on top of the Quick Seal tubes so there is no reduction of maximum radial distance and therefore no reduction of g force The shorter pathlength of the tubes also permits shorter run times For more information on the g Max system see publication DS 709 Plastic spacers have been tested for centrifugation between 2 and 25 C If spacers are centrifuged at temperatures significantly greater than 25 C deformation of the spacer and tube may occur 2 8 Preparative Ultracentrifuge Rotors and Tubes Tubes Bottles and Accessoires Table 2 2 Quick Seal Tube Spacers Part Number Spacer Description 342883 black anodized aluminum 342418 clear anodized aluminum 3
33. transparent no no no S U U U U M S S U M S propionate stainless steel opaque no no yes S U S S M S S S M S M S satisfactory resistance M z marginal resistance U unsatisfactory resistance Polyallomer polypropylene and cellulose propionate tubes with diameters of 5 to 13 mm may be sliced using the Centritube Slicer part number 347960 and appropriate adapter plate gt NOTE This information has been consolidated from a number of sources and is provided only as a guide to the selection of tube or bottle materials Soak tests at 1 g at 20 C established the data for most of the materials reactions may vary under the stress of centrifugation or with extended contact or temperature variations To prevent failure and loss of valuable sample ALWAYS TEST SOLUTIONS UNDER OPERATING CONDITIONS BEFORE USE N WARNING Do not use flammable substances in or near operating centrifuges N 1 N Preparative Ultracentrifuge Rotors and Tubes Tubes Bottles and Accessoires LABWARE MATERIAL COMPATIBILITY WITH SOLVENTS AND SAMPLE The chemical compatibility of tube or bottle materials with the gradient forming medium or other chemicals in the solution is an important consideration Although neutral sucrose and salt solutions cause no problems alkaline solutions cannot be used in Ultra Clear tubes or in polycarbonate tubes and bottles Polycarbonate and Ultra Clear tubes are incompatible with DMSO sometimes used in the prepara
34. without caps in fixed angle rotors Ultra Clear tubes made of a tough thermoplastic are thinwall and not wettable but can be made wettable see Section 3 Ultra Clear tubes are available in two types open top and Quick Seal They are transparent centrifuge tubes offering easy location of visible banded samples Standard straight wall Ultra Clear tubes must be filled completely and capped for use in fixed angle rotors Ultra Clear tubes are designed to be used one time only These tubes have good resistance to most weak acids and some weak bases but are unsatisfactory for DMSO and most organic solvents including all alcohols Ultra Clear tubes should not be autoclaved CELLULOSE PROPIONATE TUBES Cellulose propionate tubes used in some fixed angle rotors are trans parent and designed for one time use They are used without caps and should be full for centrifuging They should not be autoclaved or sterilized with alcohol These tubes have good tolerance to all gradient media including alkalines They are unsatisfactory for most acids and alcohols Preparative Ultracentrifuge Rotors and Tubes Tubes Bottles and Accessoires STAINLESS STEEL TUBES Stainless steel tubes offer excellent resistance to organic solvents and heat but should not be used with most acids or bases They offer only marginal resistance to most gradient forming materials other than sucrose and glycerol Stainless steel tubes are very strong and can be cent
35. yes yes yes yes yes no polyethylene no no yes yes yes yes yes yes yes cellulose propionate no no no no no yes yes yes no stainless steel yes yes yes yes yes no yes yes no To avoid deformation autoclave tubes or bottles open end down in a tube rack at 15 psig for no more than 20 minutes allow to cool before removing from rube rack DO NOT autoclave capped or sealed tubes or bottles Flammable do not use in or near operating ultracentrifuges 3 Do not use if there is methanol in the formula 4 Tube life will be reduced by autoclaving 5 Discoloration may occur Can be used if diluted 7 Below 26 C only 8 Below 21 C only 9 Marginal A cold sterilization method such as immersion in 10 hydrogen peroxide for 30 minutes may be used on Ultra Clear tubes Refer to Table 7 1 to select cold sterilization materials that will not damage tubes and accessories While Beckman Coulter has tested these methods and found that they do not damage the components no guarantee of sterility or disinfec tion is expressed or implied When sterilization or disinfection is a concern consult your laboratory safety officer regarding proper methods to use 7 8 Preparative Ultracentrifuge Rotors and Tubes Care and Maintenance INSPECTION Inspect containers and accessories before use hw i N ie s Inspect tubes and bottles for cracks or any major deformities before using them inh e Do not use
36. 0 0 0 0 eee eee eee 6 4 Prerun Safety Checks 0 0 0 e eee 6 4 Rotor Preparation and Loading 0 00005 6 5 ODeratiORD doce sede do edo iae terr o Puit Gea EO 4 Donee cs den 6 7 Installing the Rotor 0 0 0 ee eee eee A 6 8 Removal and Sample Recovery 000002 e eae 6 8 CARE AND MAINTENANCE Rotor Cate eee ee inem eren e e e Noah oe 7 1 Cleaning 22m sanded ave boa deed ae siardagade 7 2 Decontamination scssi ereot ns cece eee eee nee 7 3 Sterilization and Disinfection 0 000000 ee eee 7 4 INSPECHON ius seed dee ede he oe os 7 4 Field Rotor Inspection Program 00 0 0 00 ee eee 7 5 Dubtication eed eve LE e Le ra c e e VER Lael we 7 5 Overspeed Disk Replacement 0 000000 00 ee 7 6 Tube Bottle and Accessory Care 00 cece eee ee 7 6 Cleaning 2 sia cea teat es dha sed RE dag bide heut net 7 6 Decontamination 0 cece een eens 7 7 Sterilization and Disinfection 0 0 0 0 00002 a ee 7 7 Inspection 4 2 00 theese eka ee bate tio bee dee hee dad 7 9 Tube and Bottle Storage 0 0 ee eee eee ee 7 9 Removing Jammed or Collapsed Tubes 7 9 Preparative Ultracentrifuge Rotors and Tubes Page Tube Cap Cate iacob oe e ET E CR Uca EEEE 7 10 Cleaning 2 424 222 2 eae Bos RR EDI Eph de dd 7 10 Decontamination 0 0 0 cece eee eh 7 11 Sterilization and Disinfection 0 02000 e eee 7 1
37. 1 1 Beckman Coulter Preparative Rotors by Use continued Relative Number of Nominal Maximum Centrifugal Tubes x Nominal Rotor For Use in Speed Field x g k Capacity mL Capacity Instruments Rotor rpm at Imax Factor of Largest Tube mL Classified Rotors for Centrifuging Large Particles Type 70 1 Ti 70 000 450 000 36 12 x 13 5 162 Gs H R S Type 50 50 000 196 000 65 10x10 100 A B C D F G H Q R S Type 40 40 000 145 000 122 12 x 13 5 162 A B C D F GH Q R S Type 30 30 000 106 000 213 12 x 38 5 462 H R S Rotors for Centrifuging Large Particles in Volume Type 21 21 000 60 000 402 10 x 94 940 H R S Type 19 19 000 53 900 951 6 x 250 1500 H R S Type 16 16 000 39 300 1350 6 x 250 1500 H R S Rotors for Isopycnic and Rate Zonal Gradients SW 65 Ti 65 000 421 000 46 3x5 15 Gs H R S SW 60 Ti 60 000 485 000 45 6x4 24 Gs H R S SW 55 Ti 55 000 368 000 48 6x5 30 Gs H R S SW 50 1 50 000 300 000 59 6x5 30 A B C D F G H Q R S Rotors with Long Slender Tubes for Rate Zonal Gradients SW 41 Ti 41 000 288 000 124 6 x 13 2 79 2 C D F G H R S SW 40 Ti 40 000 285 000 137 6 x 14 84 G H R S SW 32 Ti 32 000 175 000 204 6 x 38 5 231 H R S SW 28 18 28 000 150 000 276 6 x 17 102 C D E GH R S Rotors for Larger Volume Density Gradients SW 32 1 32 000 187 000 228 6 x 17 102 H R S SW 30 1 30 000 124 000 138 6x8 48 B C D E GH
38. 1 8 0 308 1 391 1 3709 38 528 6 3 140 1 0447 1 3372 6 62 8 0 373 1 406 1 3722 39 548 3 3 257 1 0531 1 3380 7 73 7 0 438 1 4196 1 3735 40 567 8 3 372 1 0615 1 3388 8 84 9 0 504 1 435 1 3750 41 588 4 3 495 1 0700 1 3397 9 96 3 0 572 1 450 1 3764 42 609 0 3 617 1 0788 1 3405 10 107 9 0 641 1 465 1 3778 43 630 0 3 742 1 0877 1 3414 11 119 6 0 710 1 481 1 3792 44 651 6 3 870 1 0967 1 3423 12 131 6 0 782 1 4969 1 3807 45 673 6 4 001 1 1059 1 3432 13 143 8 0 854 1 513 1 3822 46 696 0 4 134 1 1151 1 3441 14 156 1 0 927 1 529 1 3837 47 718 6 4 268 1 1245 1 3450 15 168 7 1 002 1 546 1 3852 48 742 1 4 408 1 1340 1 3459 16 181 4 1 077 1 564 1 3868 49 766 4 4 552 1 1437 1 3468 17 194 4 1 155 1 5825 1 3885 50 791 3 4 700 1 1536 1 3478 18 207 6 1 233 1 601 1 3903 51 816 5 4 849 1 1637 1 3488 19 221 1 1 313 1 619 1 3920 52 841 9 5 000 1 1739 1 3498 20 234 8 1 395 1 638 1 3937 53 868 1 5 156 1 1843 1 3508 21 248 7 1 477 1 658 1 3955 54 859 3 5 317 1 1948 1 3518 22 262 9 1 561 1 6778 1 3973 55 922 8 5 481 1 2055 1 3529 23 277 3 1 647 1 699 1 3992 56 951 4 5 651 1 2164 1 3539 24 291 9 1 734 1 720 1 4012 57 980 4 5 823 1 2275 1 3550 25 306 9 1 823 1 741 1 4032 58 1009 8 5 998 1 2387 1 3561 26 322 1 1 913 1 763 1 4052 59 1040 2 6 178 1 2502 1 3572 27 337 6 2 005 1 7846 1 4072 60 1070 8 6 360 1 2619 1 3584 28 353 3 2 098 1 808 1 4093 61 1102 9 6 550 1 2738 1 3596 29 369 4 2 194 1 831 1 4115 62 1135 8 6 746 1 2858 1 3607 30 385 7 2 291 1 856 1 4137 63 1167 3 6 945 1 298 1
39. 2 Lubtication esae eR ere om Wa eee BA a ee ek arent ae 7 12 Inspection szbveppebebosvebbwespebbew4 ak ee 7 13 Nylon Insert Replacement 0 00 eee eee 7 13 Returning a Rotor or Accessory to the Factory 7 14 Diagnostic Hints 2 0 keene 7 15 Appendix A Chemical Resistances for Beckman Coulter Centrifugation Products A 1 Appendix B Use of the ot Integrator B 1 Appendix C The Use of Cesium Chloride Curves C 1 Appendix D Gradient Materials D 1 Appendix E Glossary of Terms E 1 Appendix F References 0 000 e cece eens F 1 Warranty Preparative Ultracentrifuge Rotors and Tubes Contents vii Illustrations Page Figure 1 1 Fixed Angle Swinging Bucket Vertical Tube and Near Vertical lube ROOTS act ede oer Ua eye eed eae Ro edenda e 1 2 Figure 1 2 Particle Separation in Fixed Angle Swinging Bucket Vertical Tube and Near Vertical Tube Rotors lessen 1 5 Figure 1 3 Sedimentation Coefficients in Svedberg Units for Some Common Biological Materials 0 0 0 eee eee eee 1 9 Figure 1 4 Nomogram 0 c cece eee een ees 1 10 Figure 1 5 Arranging Tubes Symmetrically ina Rotor 1 14 Figure 3 1 Filling OptiSeal Tubes 0 0 0 eee eee eee 3 5 Figure 3 2 The Cordless Quick Seal Tube Topper 0 0008 3
40. 3 1 22421 1 24059 1 24257 1 2542 1 2146 28 1 26414 1 27435 1 27395 1 24524 1 26380 1 26616 1 2782 1 2346 30 1 28817 1 29973 1 29944 1 26691 1 28784 1 29061 1 3028 1 2552 35 1 35218 1 36764 1 36776 1 82407 1 35191 1 35598 1 3281 1 2764 40 1 42245 1 44275 1 44354 1 88599 1 42233 1 42806 45 1 49993 1 52626 1 52803 1 45330 1 50010 1 50792 50 1 58575 1 61970 1 62278 1 52675 1 58639 1 59691 55 1 68137 1 72492 1 68254 1 69667 60 1 78859 1 80924 65 1 90966 1 93722 Preparative Ultracentrifuge Rotors and Tubes angular velocity anodized coating autoclaving buoyant density Buna N centrifugal effect centrifugal force Preparative Ultracentrifuge Rotors and Tubes Appendix E Glossary of Terms rate of rotation measured in radians per second PR 2nrpm 60 Or 0 10472 rpm a thin hard layer of aluminum oxide formed electrochemically on aluminum rotor and or accessory surfaces as a protective coating for corrosion resistance sterilization by heat dry or steam the density of a particle in a specified liquid medium black nitrile rubber used for O rings and gaskets in rotor assemblies should be used at temperatures between 34 and 121 C 30 and 250 F accumulated value of t2 foar ti where f is time and is angular velocity in a centrifugal field the force which causes a particle to move away from the center
41. 3 5 108 H R S VTi 80 80 000 510 000 8 8x 5 1 40 8 H R S Type 75 Ti 75 000 502 000 35 8 x 13 5 108 G5 H R S NVT 65 2 65 000 416 000 16 16x 5 1 81 6 H R S NVT 65 65 000 402 000 21 8 x 13 5 108 H R S VTi 65 2 65 000 416 000 10 16 x 5 1 81 6 H R S VTi 65 1 65 000 402 000 13 8 x 13 5 108 H R S VTi 65 65 000 404 000 10 8x 5 1 40 8 H R S Type 65 65 000 368 000 45 8 x 13 5 108 GS H R S Type 50 Ti 50 000 226 000 78 12 x 13 5 162 GS H R S Rotors for Centrifuging Small Particles in Volume Type 70 Ti 70 000 504 000 44 8 x 38 5 308 G5H R S Type 60 Ti 60 000 362 000 63 8 x 38 5 308 G5H R S Type 55 2 Ti 55000 340 000 64 10 x 38 5 385 Gs H R S VC 53 53 000 249 000 36 8 x 39 312 H R S Type 50 2 Ti 50 000 302 000 69 12 x 39 468 F GSH R S VAC 50 50 000 242 000 36 10 x 39 390 H R S VTi 50 50 000 242 000 36 8 x 39 312 H R S Type 45 Ti 45 000 235 000 133 6 x 94 564 F GsH Q R S Type 42 1 42 000 195 000 133 8 x 38 5 308 H R S Type 35 35 000 143 000 225 6 x 94 564 H R S Type 28 28 000 94 800 393 8 x 40 320 H4R S Rotors for Differential Flotation Type 50 4 Ti 50 000 312 000 33 44 x 6 5 286 Gs H R S Type 50 3 Ti 50000 223 000 49 18 x 6 5 117 B C D F G H Q R S Type 42 2 Ti 42 000 223 000 12 72 x 230 uL 16 5 Gs H R S Type 25 25 000 92 500 62 100 x 1 100 C D EGHR S Continued 1 6 Preparative Ultracentrifuge Rotors and Tubes Rotors Table
42. 3376 16 23 8 66 46 25 6 93 303823 13 08 8 66 46 25 6 93 303699 13 08 8 66 46 25 33 91 3 303401 16 23 13 34 26 97 44 73 303956 16 23 13 34 26 97 31 50 3 5 350781 38 25 11 10 71 12 14 30 4 303402 16 23 13 34 36 50 35 20 303957 16 23 13 34 35 50 22 23 6 5 303313 16 23 13 34 58 72 12 98 303392 25 65 13 34 58 72 25 40 303449 38 23 13 34 58 72 37 31 303687 25 65 13 34 69 85 11 13 10 5 303459 38 23 13 34 84 12 11 91 13 5 303307 25 65 16 51 71 42 12 70 303448 38 23 16 51 71 42 24 61 3 Using Tubes Bottles 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 bottles and accessories that can be used in a particular rotor Rotor use instructions are in Section 4 for fixed angle rotors in Section 5 for swinging bucket rotors and in Section 6 for vertical tube and near vertical tube rotors A table of chemical resistances is in Appendix A of this manual Reference information on some commonly used gradient materials is in Appendix D GRADIENT PREPARATION Many commercial gradient formers are available These devices usually load a tube by allowing the gradient solutions to run down the m side of the tube The heaviest concentration is loaded first followed First 5 by successi
43. 355874 858121 976959 10to 11 N m 90 to 100 in Ib VC53 355587 889096 355588 17 5 Nem 150 in Ib VTi50 355587 889096 355588 17 5 Nem 150 in lb VAC 50 355587 889096 355588 17 5 Nem 150 in Ib Part number 858121 is a 4 in drive torque wrench part number 889096 is a 8 in drive torque wrench The VTi 50 and VC 53 rotors and rotor plugs must be cooled or warmed to operating temperature before torquing or leakage may result AN CAUTION The VC 53 and VTi 50 rotors and rotor plugs must be cooled or warmed to the operating temperature prior to torquing or leakage may occur 7 Remove the rotor from the vise For runs at other than room temperature refrigerate or warm the rotor beforehand for fast equilibration 6 7 Using Vertical Tube and Near Vertical Tube Rotors INSTALLING THE ROTOR Lower the rotor straight down 1 onto the drive hub Carefully lower the rotor straight down onto the drive hub Careful installation will prevent disturbing the sample or tripping the imbalance detector Refer to the centrifuge instruction manual for detailed operating information REMOVAL AND SAMPLE RECOVERY N CAUTION 1 2 3 4 5 Tube Removal Tool 6 361668 6 8 If disassembly reveals evidence of leakage you should assume that some fluid escaped the rotor Apply appropriate decontamination procedures to the centrifuge and accessor
44. 3619 31 402 4 2 390 1 880 1 4160 64 1203 2 7 146 1 311 1 3631 32 419 5 2 492 1 9052 1 4183 65 1238 4 7 355 1 324 1 3644 33 436 9 2 595 Computed from the relationship p 10 2402 nD 5 12 6483 for densities between 1 00 and 1 37 and p 5 10 8601 nD25 13 4974 for densities above 1 37 Bruner and Vinograd 1965 Divide by 10 0 to obtain 96 w v Density data are from International Critical Tables Preparative Ultracentrifuge Rotors and Tubes Gradient Materials Table D 3 Density Refractive Index and Concentration Data Sucrose at 20 C Molecular Weight 342 3 Density Refractive 96 by mg mL of Density Refractive by mg mL of g cm3 Index nD Weight Solution Molarity g cm3 Index nD Weight Solution Molarity 0 9982 1 3330 0 1 1463 1 3883 34 389 7 1 138 1 0021 1 3344 1 10 0 0 029 1 1513 1 3902 35 403 0 1 177 1 0060 1 3359 2 20 1 0 059 1 1562 1 3920 36 416 2 1 216 1 0099 1 3374 3 30 3 0 089 1 1612 1 3939 37 429 6 1 255 1 0139 1 3388 4 40 6 0 119 1 1663 1 3958 38 443 2 1 295 1 0179 1 3403 5 50 9 0 149 1 1713 1 3978 39 456 8 1 334 1 0219 1 3418 6 61 3 0 179 1 1764 1 3997 40 470 6 1 375 1 0259 1 3433 7 71 8 0 210 1 1816 1 4016 41 484 5 1 415 1 0299 1 3448 8 82 4 0 211 1 1868 1 4036 42 498 5 1 456 1 0340 1 3464 9 93 1 0 272 1 1920 1 4056 43 512 6 1 498 1 0381 1 3479 10 103 8 0 303 1 1972 1 4076 44 526 8 1 539 1 0423 1 3494 11 114 7 0 335 1 2025 1 4096 4
45. 3624 8 mm 5 16 in hand tighten 303658 303379 Hex driver 841883 303113 346256 11 mm 7 16 in hand tighten 305022 330860 301870 338907 303319 302359 326891 302133 326905 338903 337927 Unless otherwise indicated caps are clear anodized aluminum Unless otherwise indicated cap nuts are aluminum 3 Slide the tube up around the stem PAST the O ring or gasket as shown in Figure 3 4 slightly rotating the cap assembly The tube wall should pass between the O ring or gasket and the crown so that the top of the tube rests on the underside of the crown Tighten the nut by hand just enough to hold the tube cap in place 4 Position the capped tube in the appropriate sized hole from the underside of the tube cap vise 305075 The vise must be correctly mounted to the bench with the clamping positioned on the right see Figure 3 5 or crimping of the crown may result While holding the tube with one hand tighten the vise around the crown by using the clamping knob Make sure that the cap and the tube are level horizontal 5 Tighten the cap nut as described in Table 3 4 6 Usea syringe to finish filling the tube through the filling hole in the stem e Thinwall tubes must be as full as possible to prevent tube collapse Preparative Ultracentrifuge Rotors and Tubes 3 21 Using Tubes Bottles and Accessories CORRECT WRONG O ring Tube below Tube beyond the O ring the O ring the
46. 4 302312 801761 326899 SS 45 Ti 35 21 330901 330791 330793 338864 302312 346242 330900 thinwall PA 45 Ti 35 UC 3389054 330791 338913f 338864 302312 341767 338909 thickwall PA 45 Ti 35 PC a Tube caps are aluminum unless otherwise noted b Abbreviations PA polyallomer PC polycarbonate SS stainless steel UC UltraClear Aluminum and stainless steel 4 Tube cap is optional Use a tube cap when centrifuging a thickwall tube at its maximum fill capacity Titanium f Washer part number 330899 is also required Aluminum and t itanium Preparative Ultracentrifuge Rotors and Tubes Using Tubes Bottles and Accessories Aluminum Caps Aluminum caps are anodized for corrosion resistance with colored crowns for identification Red anodized Aluminum caps aluminum stem and crown with red anodized crowns are used with thinwall Ultra Clear and polyallomer tubes in high performance rotors These extra strength caps are designed for the greater forces generated in the high performance rotors The cap nut should be tightened with a torque wrench while the tube is held in the tube cap vise Blue anodized Aluminum caps with blue anodized crowns are used with thickwall polyallomer and polycarbonate tubes for centrifuga tion at their maximum fill volumes in high performance rotors The cap nut should be tightened with a torque wrench while the tube is
47. 42696 clear anodized aluminum 342695 red anodized aluminum 342699 red anodized aluminum p 342417 clear anodized aluminum 342697 titanium ZEE 344389 white Delrin 344634 white Delrin 344635 344676 black Noryl p 345828 black Noryl EE 349289 blue anodized aluminum 358164 black Delrin SS Preparative Ultracentrifuge Rotors and Tubes Tubes Bottles and Accessories ADAPTERS hd Adapters Delrin Adapters Many rotors can accommodate a variety of tube sizes by using adapters that line the tube cavity or bucket Small open top tubes use Delrin adapters which line the tube cavity or bucket Adapters with conical cavities must be used to support both open top and Quick Seal konical tubes Tubes used with adapters can be filled and capped according to the type of tube and the design of the rotor being used Many of the small straightwall tubes when used with adapters require speed reductions due to the added density of Delrin 1 4 g mL Additional speed reductions for heavy tube loads may also be required refer to ALLOWABLE RUN SPEEDS in Section 1 In vertical tube rotors fmin is unchanged see the illustration in Figure 1 2 However in fixed angle and near vertical tube rotors r must be calculated rnin TE 1 sinQ cosO L sind 9 where Tmax the distance in millimeters from the axis of rotation to the farthest part of the tube cavity diameter o
48. 5 Quick Seal Tubes irs reo RR e ee ce 3 28 Making Ultra Clear Tubes Wettable 0 3 29 USING FIXED ANGLE ROTORS DESCrIPtiOn i cure eet Een RR Ue RR eos Rb E 4 1 Tubes and Bottles saneta eR RR RUE EAR 4 4 Rotor Preparation and Loading 0 0 0c cee eee ee 4 4 Prerun Safety Checks 0 0 0 cece cece eee eee 4 6 Rotor Preparation and Loading 00 0 5 4 6 ocu MPH m 4 7 Installing the Rotor seeeeeeee RA 4 8 Removal and Sample Recovery 00 00 eee eee 4 8 USING SWINGING BUCKET ROTORS D scrptlOB cor be hae aes eee Ree A EE RR rep Rp EE 5 1 Tubes and Bottles 4 24 nc 088 Meade SYR eee See 5 4 Rotor Preparation and Loading 0 00 e eee eee 5 4 Prerun Safety Checks 0 0 0 0 c cece eee eee 5 4 Rotor Preparation and Loading 004 5 5 Operation wo ethane S uet ede E race de e aia as 5 6 Installing the Rotor ssseeeelee IA 5 6 Removal and Sample Recovery 0 00 eee eee 5 8 Preparative Ultracentrifuge Rotors and Tubes Contents Contents vi SECTION 6 SECTION 7 Page USING VERTICAL TUBE AND NEAR VERTICAL TUBE ROTORS DesctiptiOll ies ta terns bb duck boe e oS Ree eee eee ba ds 6 1 Vertical Tube Rotors 0 0 0 eee ee eee 6 1 Near Vertical Tube Rotors 0 0 0 0c eee ee eee eee 6 2 Tubes and Bottles 0 ce eee eee 6 4 Rotor Preparation and Loading
49. 5 541 1 1 581 1 0465 1 3510 12 125 6 0 367 1 2079 1 4117 46 555 6 1 623 1 0507 1 3526 13 136 6 0 399 1 2132 1 4137 47 570 2 1 666 1 0549 1 3541 14 147 7 0 431 1 2186 1 4158 48 584 9 1 709 1 0592 1 3557 15 158 9 0 464 1 2241 1 4179 49 599 8 1 752 1 0635 1 3573 16 170 2 0 497 1 2296 1 4200 50 614 8 1 796 1 0678 1 3590 17 181 5 0 530 1 2351 1 4221 51 629 9 1 840 1 0721 1 3606 18 193 0 0 564 1 2406 1 4242 52 645 1 1 885 1 0765 1 3622 19 204 5 0 597 1 2462 1 4264 53 660 5 1 930 1 0810 1 3639 20 216 2 0 632 1 2519 1 4285 54 676 0 1 975 1 0854 1 3655 21 227 9 0 666 1 2575 1 5307 55 691 6 2 020 1 0899 1 3672 22 239 8 0 701 1 2632 1 4329 56 707 4 2 067 1 0944 1 3689 23 251 7 0 735 1 2690 1 4351 57 723 3 2 113 1 0990 1 3706 24 263 8 0 771 1 2748 1 4373 58 739 4 2 160 1 1036 1 3723 25 275 9 0 806 1 2806 1 4396 59 755 6 2 207 1 1082 1 3740 26 288 1 0 842 1 2865 1 4418 60 771 9 2 255 1 1128 1 3758 27 300 5 0 878 1 2924 1 4441 62 788 3 2 303 1 1175 1 3775 28 312 9 0 914 1 2983 1 4464 62 804 9 2 351 1 1222 1 3793 29 325 4 0 951 1 3043 1 4486 63 821 7 2 401 1 1270 1 3811 30 338 1 0 988 1 3103 1 4509 64 838 6 2 450 1 1318 1 3829 31 350 9 1 025 1 3163 1 4532 65 855 6 2 500 1 1366 1 3847 32 363 7 1 063 1 3224 1 4558 66 872 8 2 550 1 1415 1 3865 33 376 7 1 100 1 3286 1 4581 67 890 2 2 864 Divide by 10 0 to obtain w v Density and refractive index data are from the International Critical Tables D 4 Preparative Ultracentrif
50. 6 8 64 10 x 38 5 50 4 Ti 50 000 312 000 20 111 5 96 2 80 8 33 44 x 6 5 50 3 Ti 50 000 223 000 20 79 5 64 2 48 9 49 18x 6 5 50 2 Ti 50 000 302 000 24 107 9 81 2 54 4 69 12x 39 50 Ti 50 000 226 000 26 80 8 59 1 37 4 78 12 x 13 5 50 50 000 196 000 20 70 1 53 6 37 0 65 10x10 45 Ti 45 000 235 000 24 103 8 69 8 35 9 133 6x94 42 1 42 000 195 000 30 98 6 68 8 39 1 133 8 x 38 5 42 2 Ti 42 000 223 000 30 113 0 108 5 104 0 9 72 x 230 uL 40 3 40 000 142 000 20 79 5 64 2 48 9 77 18x 6 5 40 40 000 145 000 26 80 8 59 1 37 4 122 12 x 13 5 35 35 000 143 000 25 104 0 69 5 35 0 225 6 x 94 30 30 000 106 000 26 104 8 77 0 49 1 213 12 x 38 5 28 28 000 94 800 34 108 0 70 0 32 0 393 8 x 50 25 25 000 92 500 25 132 1 122 8 113 4 62 100 x 1 21 21 000 60 000 18 121 5 90 9 60 3 402 10x 94 19 19 000 53 900 25 133 4 83 9 34 4 951 6 x 250 16 16 000 39 300 25 137 0 86 0 35 0 1350 6 x 250 Maximum speeds are based on a solution density of 1 2 g mL in all fixed angle rotors except for the Type 60 Ti Type 42 1 and the Type 35 which are rated for a density of 1 5 g mL Maximum RCF measured at outer row Maximum RCF measured at the third row Radial distances are those of the third row Preparative Ultracentrifuge Rotors and Tubes 4 3 Using Fixed Angle Rotors I gt NOTE Although rotor components and accessories made by other manufacturers may fit in the Beckman Coulter rotor you are using their safety in
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52. 7558 e mail beckman_uk beckman com Additional addresses are available at www beckmancoulter com 4300 N Harbor Boulevard Box 3100 Fullerton California 92834 3100 Internet www beckmancoulter com Telex 678413 Fax 1 800 643 4366 2007 Beckman Coulter Inc CA Bd Printed on recycled paper
53. 8 Figure 3 3 Tools Used to Assemble Tube Caps 20 000000 3 20 Figure 3 4 Tube Cap Installation 0 0 0 0 c cee eee 3 22 Figure 3 5 Tube Cap Vise 0 0 aies mia cee een eens 3 23 Figure 4 1 Fixed Angle Rotors 1 0 0 0 0 cece eects 4 2 Figure 4 2 Removal Tools Used in Fixed Angle Rotors 00 4 9 Figure 5 1 Swinging Bucket Rotors 0 ccc eee eee 5 2 Figure 5 2 Checking Hook on Bucket Positions After the Rotor is Installed 5 7 Figure 6 1 Vertical Tube Rotors leere 6 3 Figure 6 2 Near Vertical Tube Rotors 2 0 0 0 eee ee eA 6 3 Figure 6 3 Preparing a Vertical Tube or Near Vertical Tube Rotor 6 6 viii Preparative Ultracentrifuge Rotors and Tubes Contents Page Figure B 1 The s t Charts for the SW 60 Ti Rotor issssee esses B 3 Figure C 1 Precipitation Curves for the Type 90 Ti Rotor C 2 Figure C 2 CsCl Gradients at Equilibrium 00 0 0 e eee eee eee C 3 Preparative Ultracentrifuge Rotors and Tubes ix Tables Page Table 1 1 Beckman Coulter Preparative Rotors by Use 0 1 6 Table 2 1 Characteristics and Chemical Resistances of Tube and Bottle Materials 0 0 eee eee eee 2 2 Table 2 2 Quick Seal Tube Spacers 0 0 ee eee eee 2 9 Table 2 3 Dimensions of Delrin Adapters lees 2 11 Table 3 1 Filling and Capping Requirements for Tubes and Bottles
54. 8 rotors in Model L8 s and L8M s only Any Beckman Coulter preparative rotor EXCEPT the Type 15 rotor and zonal and continuous flow rotors Any Beckman Coulter preparative rotor including zonal and continuous flow rotors Special Action on Older Type 35 and Type 42 1 Rotors We have found that there is a high risk associated with Type 35 rotor and Type 42 1 rotors having serial numbers 1299 and lower These rotors were originally stamped Type 42 or Type 50 2 and were derated over 15 years ago THESE ROTORS ARE NOW OVER 20 YEARS OLD AND MUST BE RETIRED IMMEDIATELY REGARDLESS OF THE INSTRUMENTS IN WHICH THEY ARE USED To the maximum speed of the ultracentrifuge as applicable Preparative Ultracentrifuge Rotors and Tubes Rotors This section is an introduction to the Beckman Coulter family of preparative ultracentrifuge rotors providing general information on rotor design selection and operation Rotor designs described are fixed angle swinging bucket vertical tube and near vertical tube type Specific instructions for using each type of rotor are contained in Sections 4 through 6 Care and maintenance information for all of these rotors is contained in Section 7 Analytical continuous flow and zonal rotors are not covered in this manual they are described in detail in their respective rotor instruction manuals GENERAL DESCRIPTION ROTOR DESIGNATIONS Beckman Coulter preparative rotors are named according to t
55. ESCRIPTION VERTICAL TUBE ROTORS Preparative Ultracentrifuge Rotors and Tubes 6 Using Vertical Tube and Near Vertical Tube Rotors This section contains instructions for using vertical tube and near vertical tube rotors in preparative ultracentrifuges In addition to these instructions observe procedures and precautions provided in the applicable rotor and ultracentrifuge manuals Refer to Section 2 for labware selection information and Section 3 for recommended filling and sealing or capping requirements and for sample recovery procedures Refer to Section 7 for information on the care of rotors and accessories Vertical tube and near vertical tube rotors are especially useful for isopycnic banding and rate zonal experiments Some rotors have fluted bodies designed to eliminate unnecessary weight and minimize stresses Refer to Table 6 1 for general rotor specifications Tubes in vertical tube rotors see Figure 6 1 are held parallel to the axis of rotation in numbered tube cavities These rotors have plugs that are screwed into the rotor cavities over sealed OptiSeal or Quick Seal tubes The plugs with spacers when required restrain the tubes in the cavities and provide support against the hydrostatic force generated by centrifugation 6 1 Using Vertical Tube and Near Vertical Tube Rotors Table 6 1 General Specifications for Beckman Coulter Preparative Vertical Tube and Near Vertical Tube Rotors Rotors listed in paren
56. G QUICK SEAL TUBES Fill each tube to the base of the neck using a syringe with a 13 gauge or smaller needle A small air space no larger than 3 mm may be left but an air bubble that is too large can cause the tube to deform disrupting gradients or sample Spacer and or floating spacer require ments for Quick Seal tubes are described in the individual rotor manuals The neck of the tube should be clean and dry before sealing There are two tube sealers for use with Quick Seal tubes the hand held Cordless Tube Topper and the older tabletop model no longer available Refer to How to Use Quick Seal9 Tubes with the Beckman Cordless Tube Topper M publication IN 181 for detailed information about the Tube Topper Instructions for using the older tabletop tube sealer are in How to Use Quick Seal Tubes with the Beckman Tube Sealer publication IN 163 Quick Seal tubes are heat sealed quickly and easily using the Beckman Cordless Tube Topper see Figure 3 2 The following procedures provide the two methods for heat sealing Quick Seal tubes using the hand held Tube Topper Use the applicable tube rack listed in the appropriate rotor manual Charging Stand Figure 3 2 The Cordless Quick Seal Tube Topper 3 A sample application block 342694 is available for holding and compressing tubes and can be used to layer samples on preformed gradients in polyallomer Quick Seal tubes 3 8 Preparative Ultracentrifuge Rotors and
57. If the sample is to be layered on top be sure to allow enough room for the sample so that there is no fluid in the tube stem Figure 3 1 Filling OptiSeal Tubes Stems are large enough to accept standard pipettes Preparative Ultracentrifuge Rotors and Tubes 3 5 Using Tubes Bottles and Accessories Table 3 2 OptiSeal Tubes and Accessories Spacers are shown in the correct orientation for placement onto tubes Size Volume Part Number Rack mm mL pkg 56 Spacer Assembly Rotor 361627 361678 pkg 2 w amber Ultem SW 55 Ti 13 x 33 3 3 m 361650 SW 50 1 361621 Bell top 361676 pkg 2 amber Ultem wu A uy Type 50 4 Ti 13 x 48 4 7 C 361638 Type 50 3 Ti 362185 362198 VTi 90 VTi 80 gold aluminum VTi 65 2 NVT 361638 90 13 x 51 4 9 NVT 65 2 362199 a black Noryl 361638 VTi 65 361623 Bell top 361670 pkg 2 Type 90 Ti amber Ultem Type 80 Ti Type 70 1 Ti 16 x 60 8 9 361642 Type 65 Type 50 Ti Type 50 362181 362202 gold aluminum NVT 65 900998 VTI 65 1 16x70 11 2 m 361646 361625 Bell top Type 50 2 Ti Type 42 1 wm Type 30 25 x 77 32 4 m 392833 pkg 2 amber Ultem SW 32 Ti 361646 SW 28 362183 362204 gold aluminum VTi 50 wa 25 x 89 36 2 360542 VAC 50 VC 53 Disposable plastic plugs included 361669 pkg 2 Type 70 Ti amber Ultem Type 60 Ti Type 55 2 Ti
58. LR IM 24 COULTER ROTORS AND TUBES For Beckman Coulter Preparative Ultracentrifuges User s Manual SAFETY NOTICE This safety notice summarizes information basic to the safe operation of the rotors and accessories described in this manual The international symbol displayed above is a reminder that all safety instructions should be read and understood before use or mainte nance of rotors or accessories When you see the symbol on other pages pay special attention to the safety information presented Also observe any safety information contained in applicable rotor and instrument manuals Observance of safety precautions will help to avoid actions that could cause personal injury as well as damage or adversely affect the performance of the instrument rotor tube system Chemical And Biological Safety Normal operation may involve the use of solutions and test samples that are pathogenic toxic or radioactive Such materials should not be used in these rotors however unless all necessary safety precautions are taken Observe all cautionary information printed on the original solution containers prior to their use Handle body fluids with care because they can transmit disease No known test offers complete assurance that body fluids are free of micro organisms Some of the most virulent Hepatitis B and C and HIV I V viruses atypical mycobacterium and certain systemic fungi furthe
59. R S SW 30 30 000 124 000 138 6 x 20 120 B C D F G H R S SW 288 28 000 141 000 245 6 x 38 5 231 C D F G H R S SW 25 1 25 000 90 400 337 3 x 34 102 A B C D F G H Q R S a Maximum speeds are based on a solution density of 1 2 g mL in all rotors except for the Type 60 Ti Type 42 1 and the Type 35 which are rated for a density of 1 5 g mL and the near vertical tube and vertical tube rotors which are rated for a density of 1 7 g mL b Relative Centrifugal Field RCF is the ratio of the centrifugal acceleration at a specified radius and speed r to the standard acceleration of gravity g according to the following formula RCF rw g where r is the radius in millimeters cis the angular velocity in radians per second 2mRPM 60 and g is the standard acceleration of gravity 9807 mm s After substitution RCF 1 12r RPM 100 Class G Model L3 only 4 Except L5 and LSB Maximum RCF measured at outer row f Maximum RCF measured at the third outermost row Radial distances are those of the third row g SW 28 1M and SW 28M rotors no longer manufactured are specially modified versions of the SW 28 1 and SW 28 rotors and are equipped with a mechanical overspeed system These rotors are otherwise identical to the SW 28 1 and SW 28 rotors Preparative Ultracentrifuge Rotors and Tubes 1 7 Rotors PELLETING DIFFERENTIAL SEPARATION Pelleting separates particles of different sedimentation coeffi
60. REMOVAL AND SAMPLE RECOVERY 4 8 N CAUTION If disassembly reveals evidence of leakage you should assume that some fluid escaped the rotor Apply appropriate decontamination procedures to the centrifuge and accessories Remove the rotor from the centrifuge by lifting it straight up and off the drive hub Unscrew the handle counterclockwise and remove the lid Some rotor handles have holes so that a screwdriver or metal rod can be used to loosen the lid Remove spacers and or floating spacers with a removal tool 338765 or hemostat Remove tubes or bottles from the rotor using one of the following procedures Refer to Figure 4 2 for removal tools gt NOTE When removing a tube cap do not remove the cap nut or the stem may drop into the tube contents and disturb the separation Instead loosen the nut just enough to remove the cap assembly as a unit Preparative Ultracentrifuge Rotors and Tubes For Capped Tubes For Noryl Caps 301875 335381 ye d For Noryl Floating Spacers and OptiSeal Spacers 338765 zn For Quick Seal and OptiSeal Tubes 361668 For Delrin Adapters 303419 For Aluminum Caps 878133 Figure 4 2 Removal Tools Used in Fixed Angle Rotors Extract capped tubes using the appropriate removal tool Insert the threaded end of the tool into the cap and screw at least one turn If neces
61. Seal tubes Refer to Section 6 for specific information about the use of near vertical tube rotors Table 1 1 lists Beckman Coulter preparative rotors by use 1 4 Preparative Ultracentrifuge Rotors and Tubes Rotors At Speed At Rest in Rotor At Rest Outside Rotor Fixed Angle Rotors Pathlength Swinging Bucket Rotors Pathlength Vertical Tube Rotors Tmin max V Pathlength Near Vertical Tube Rotors min max Pathlength Figure 1 2 Particle Separation in Fixed Angle Swinging Bucket Vertical Tube and Near Vertical Tube Rotors Dark gray represents pelleted material light gray is floating components and bands are indicated by black lines Preparative Ultracentrifuge Rotors and Tubes 1 5 Rotors Table 1 1 Beckman Coulter Preparative Rotors by Use Rotors listed in parentheses are no longer manufactured Relative Number of Nominal Maximum Centrifugal Tubes x Nominal Rotor For Use in Speed Field x g k Capacity mL Capacity Instruments Rotor rpm at Imax Factor of Largest Tube mL Classified Rotors for Centrifuging Extremely Small Particles NVT 100 100 000 750 000 8 8x 5 1 40 8 R S Type 100 Ti 100 000 802 400 15 8 x 6 8 54 R S NVT 90 90 000 645 000 10 8x 5 1 40 8 H R S Type 90 Ti 90 000 694 000 25 8 x 13 5 108 H R S VTi 90 90 000 645 000 6 8x 5 1 40 8 H R S Type 80 Ti 80 000 602 000 28 8 x 1
62. act the nearest Beckman Coulter Sales and Service office and request a Returned Goods Authorization RGA form and packaging instructions Please include the complete rotor assembly with buckets lid handle tube cavity caps etc A SIGNED STATEMENT THAT THE ROTOR AND ACCESSO RIES ARE NON RADIOACTIVE NON PATHOGENIC NON TOXIC AND OTHERWISE SAFE TO SHIP AND HANDLE IS REQUIRED Beckman Coulter Worldwide Biomedical Research Division Offices AUSTRALIA Beckman Coulter Australia Pty Ltd Unit D 24 College St Gladesville NSW 2111 Australia Telephone 61 2 9844 6000 or toll free 1 800 060 880 Fax 61 2 9844 6096 email lifescienceaustralia beckman com CANADA Beckman Coulter Canada Inc 6755 Mississauga Road Suite 600 Mississauga Ontario Canada L5N 7Y2 Telephone 1 905 819 1234 Fax 1 905 819 1485 CHINA Beckman Coulter Inc Beijing Representative Office Unit 2005A 2006 2009 East Ocean Center Jian Guomenwai Avenue Beijing 100004 China Telephone 86 10 6515 6028 Fax 86 10 6515 6025 6515 6026 CZECH REPUBLIC Beckman Coulter Prague Radiova 1 102 27 Prague 10 Czech Republice Telephone 420 267 00 85 13 Fax 420 267 00 83 23 EASTERN EUROPE MIDDLE EAST NORTH AFRICA Beckman Coulter International S A 22 Rue Juste Olivier Case Postale 301 303 CH 1260 Nyon Switzerland Telephone 41 22 365 3707 Fax 41 22 365 0700 FRANCE Beckman Coulter France S A Paris Nord II 33
63. age the rotor or components no guarantee of sterility or disinfec tion is expressed or implied Rotors and most rotor components except those made of Noryl can be autoclaved at 121 C for up to an hour Remove the lid bucket caps or rotor plugs and place the rotor and or buckets in the autoclave upside down O rings and gaskets can be left in place on the rotor Ethanol 7096 may be used on all rotor components including those made of plastic Bleach sodium hypochlorite may be used but may cause discoloration of anodized surfaces Use the minimum immersion time for each solution per laboratory standards Frequent and thorough inspection is crucial to maintaining a rotor in good operating condition Periodically at least monthly depending on use inspect the rotor especially inside cavities and buckets for rough spots cracks pitting white powder deposits on aluminum rotors frequently aluminum oxide or heavy discoloration If any of these signs are evident do not run the rotor Contact your Beckman Coulter repre sentative for information about the Field Rotor Inspection Program and the Rotor Repair Program Regularly check the condition of O rings or gaskets and replace any that are worn or damaged Regularly check that all sealing surfaces are smooth and undamaged to ensure proper sealing 3 Flammability hazard Do not use in or near operating ultracentrifuges 74 Preparative Ultracentrifuge Rotors and T
64. an Coulter concentrated rotor cleaning solution recommended because it is a mild solution that has been tested and found effective and safe for Beckman Coulter rotors and accessories Preparative Ultracentrifuge Rotors and Tubes SpinkoteTM sucrose supernatant Svedberg unit S swinging bucket rotor Ultem vertical tube rotor Viton wettable Preparative Ultracentrifuge Rotors and Tubes Glossary of Terms Beckman Coulter lubricant for metal to metal contacts a sugar not a self forming gradient used in rate zonal separations generally used in separating RNA subcellular organelles and cell membranes the liquid above the sedimented material following centrifugation a unit of sedimentation velocity 18S 10 3 seconds a rotor in which the tubes or bottles are carried in buckets that swing up to the horizontal position during centrifugation sometimes referred to as a horizontal or swing out rotor polyetherimide PEI used in adapters covers and spacers should be used at temperatures between 29 and 204 C 20 and 400 F Ultem is a regis tered trademark of GE Plastics a rotor in which the tubes or bottles are held parallel to the axis of rotation fluorocarbon elastomer used in high temperature applications Viton is a registered trademark of E I Du Pont de Nemours amp Company tube or bottle material that water or other aqueous solution will adhere to the more wettable a tube or bottle ma
65. and accessories Sample recovery depends on the type of labware used the compo nent s isolated and the analysis desired The Beckman Coulter Universal Fraction Recovery System 343890 can be useful when recovering sample from tubes see publication L5 TB 081 The usual methods of recovering supernatants or pellets include decanting or withdrawing the gradient and scraping pellets from the tube bottom Remove tube caps carefully to avoid sample mixing If tubes will be reused scrape pellets out with a plastic or wooden tool scratches on tube interiors caused by abrasive or sharply pointed tools can result in tube failure during subsequent runs Centrifugation exerts high forces on plastic labware The effect of these forces on OptiSeal labware is compression of the tube charac terized by tube deformation that even if slight causes a decrease in internal volume OptiSeal labware is designed to contain the resulting slight pressure increase during separation as well as during normal post separation handling However a small volume 250 uL of fluid may occasionally ooze from around the plug onto the tube stem area as a plug is removed Therefore we recommend using a tissue to contain escaped fluid when extracting plug assemblies from tubes Using Tubes Bottles and Accessories 1 After centrifugation use the spacer removal tool 338765 or a hemostat to carefully remove the spacers taking care not to scratch the
66. as on the rotor The applicable rotor instruction manual specifies which cap should be used with a particular tube or bottle A tube cap assembly includes e astem anylon insert an O ring or flat gasket crown e a Delrin crown washer in red blue and black aluminum caps ahex shaped nut and astainless steel setscrew The stem supports the upper portion of the tube To provide tube support during centrifugation the stem is longer for thinwall tubes than for thickwall or metal tubes Some stems have an abraded Preparative Ultracentrifuge Rotors and Tubes Titanium Caps Preparative Ultracentrifuge Rotors and Tubes Using Tubes Bottles and Accessories surface to increase friction between the O ring and the stem minimizing rotation of the stem when the cap nut is tightened The O ring or gasket seals the cap to tube interface The crown seats on the rotor tube cavity counterbore and supports the stem and the nut during centrifugation In some high performance rotors tube caps have crown washers The washer minimizes friction which would reduce the effective tightening of the cap nut and also protects the nut and the crown After the tube has been capped tight ened and filled the setscrew is used to seal the filling hole in the stem by seating against the nylon insert Refer to Table 3 3 for detailed information about tube caps AN CAUTION Do not interchange tube caps or tube cap components even
67. at other than room temperature refrigerate or warm the rotor beforehand for fast equilibration PRERUN SAFETY CHECKS Read all safety information in the rotor manual before using the N rotor a 1 Make sure that the rotor plugs gaskets and spacers are clean and E amp show no signs of corrosion or cracking The high forces generated 90 000 rpm in these rotors can cause damaged components to fail 20 Sector 9 35553 2 Make sure the rotor is equipped with the correct overspeed disk 4 refer to Section 1 If the disk is missing or damaged replace it as described in Section 7 6 4 Preparative Ultracentrifuge Rotors and Tubes Using Vertical Tube and Near Vertical Tube Rotors Check the chemical compatibilities of all materials used Refer to Appendix A Verify that tubes and accessories being used are listed in the applicable rotor manual ROTOR PREPARATION AND LOADING Threads S Dome Top Tube Preparative Ultracentrifuge Rotors and Tubes Gasket PA e gt Plug _ Tube Spacers ERN AN gt P E s Spacer we Floating L Spacer Bell Top Tube 1 Be sure that plug threads are clean and lightly but evenly lubri cated with Spinkote lubricant 306812 If using a rotor vise set the rotor into the vise which should be bolted or clamped to a rigid surface Dry the exterior of the plugged OptiSeal or sealed Quick Seal t
68. at the O ring plug and bottle lip are dry and free of lubrication before use e O ring or gasket damaged or defective Replace the O ring or gasket e Cap not tightened sufficiently Tighten cap securely e Sealing surface of the bottle is not smooth Replace bottle Bottle too full the meniscus must be kept lower to prevent leakage Refer to the rotor manual for fill volumes and speed reductions Fill volume too low to provide tube wall support Refer to the rotor manual for fill volumes and speed reduction Moisture between the bottle and the cavity or bucket can cause the bottle to float and collapse Ensure that bottles and cavities or buckets are dry before inserting them Reagent used that attacks the bottle material Refer to Appendix A for chemical compatibilities of bottle material and chemicals Bottles may crack or become brittle if they are used below their lower temperature limit Before using bottles at other than stated tempera ture limits evaluate them under centrifugation conditions If sample is frozen in bottles make sure that bottles are thawed to at least 22C before centrifugation Bottles may become brittle with age and use Dispose of brittle or cracked bottles Improper cleaning decontamination or sterilization procedures used Refer to Table 7 1 for acceptable procedures and materials Appendix A Chemical Resistances for Beckman Coulter Centrifugation Products To Close Rotors and T
69. ation A wet or greased O ring or gasket may allow the stem to rotate when the cap nut is tightened preventing proper sealing of the cap 6 Flammability hazard Do not use in or near operating ultracentrifuges 7 12 Preparative Ultracentrifuge Rotors and Tubes Care and Maintenance INSPECTION Inspect tube cap components before each use Refer to Table 3 4 in Section 3 of this manual for a list of cap components Inspect Here e Carefully inspect the crown for deformed or roughened edges Run the crown and the base of the O ring groove for fine circular lines or stress cracks Do not use damaged wrenches or hex drivers or Tube Cap Crown tools that have burrs A burred tool can score the crown Discard a damaged crown as it may fail and damage the rotor PA your finger around the bottom edge of the crown surfaces should Ky be flat squared off and not rounded or jagged Check the top of Inspect the cap stem for evidence of stress cracking Also make sure that threads are in good condition and properly lubricated before use Look at the underside of the stem the white nylon insert should not protrude below the filling hole If it does replace the nylon insert see replacement procedures below Inspect Here A e qp Tube Cap Stem s g Check the O ring or gasket for cuts excessive abrasions or flattened areas It is good practice to replace the O ring or gasket frequently Oncaps with filling holes inspect the
70. ation process GENERAL OPERATING INFORMATION Careful centrifugation technique is essential because forces gener ated in high speed centrifugation can be enormous For example 1 gram at the bottom of an SW 60 Ti rotor bucket rotating at 60 000 rpm exerts the gravitational equivalent of 0 5 ton of centrifugal mass at the bottom of the bucket Note the classification letter of the ultracentrifuge to be used and be sure the rotor is appropriate for the instrument see the CLASSI FICATION PROGRAM chart at the beginning of this manual and Table 1 1 Acceptable classification letters are engraved on rotor lids handles stands or bodies gt NOTE Specific information about filling sealing and capping containers loading rotors etc can be found in later sections ROTOR BALANCE The mass of a properly loaded rotor will be evenly distributed on the ultracentrifuge drive hub causing the rotor to turn smoothly with the drive An improperly loaded rotor will be unbalanced consistent running of unbalanced rotors will reduce ultracentrifuge drive life To balance the rotor load fill all opposing tubes to the same level with liquid of the same density Weight of opposing tubes must be distributed equally Place tubes in the rotor symmetrically as illus trated in Figure 1 5 Preparative Ultracentrifuge Rotors and Tubes 1 13 Rotors OVERSPEED PROTECTION e Qu X O O o Figure 1 5 Arranging Tubes Symmetrically in a Roto
71. be Contact your Beckman Coulter Service representative if you are unsuccessful A CAUTION Do not use a hemostat or any metal tool to pry a jammed or collapsed tube out of the rotor The rotor can be scratched and damaged If an uncapped polycarbonate tube is stuck remove tube contents and place the rotor or bucket upside down in an autoclave for about 30 to 60 minutes When the rotor is cool enough to handle try to remove the jammed or collapsed tube Do not autoclave sealed or capped tubes or bottles e Pour a solvent in the tube to make the tube material more flexible Several changes of solvent may be necessary to weaken the tube for easy removal Refer to the chemical resistances list in Appendix A to select a solvent that will not damage the rotor Itis very important to keep tube cap assemblies together as a unit Do NOT interchange cap components caps are designed as a unit for a particular tube being centrifuged in a particular rotor If cap compo nents are separated for cleaning be sure components are classified according to the tube and rotor for which they are designed Do not store O rings or gaskets under compression 1 Disassemble tube caps and wash them in a mild detergent solution such as Beckman Solution 555 339555 diluted 10 to 1 with water If necessary scrub the inside of caps using a cotton tipped swab or a brush that will not scratch the surface Preparative Ultracentrifuge Rotors and Tubes Care and
72. be and the bucket may lead to tube collapse and increase the force required to extract the tube Slide the filled and sealed tubes into the buckets Loaded buckets can be supported in the bucket holder rack avail able for each rotor 5 Use the required spacers and or floating spacers if necessary to complete the loading operation If OptiSeal tubes are being used install a spacer over each plugged tube refer to the applicable rotor manual Leave buckets without tubes completely empty If Quick Seal tubes are being used install spacers and or floating spacers over sealed tubes refer to the applicable rotor manual The particular type of tube support for Quick Seal tubes in swinging bucket rotors depends on the length of the tube but the top of the tube must be supported Leave buckets without tubes completely empty 6 Match numbered caps with numbered buckets Screw the caps into the bucket until there is metal to metal contact Tighten flat caps with a screwdriver 5 5 Using Swinging Bucket Rotors I gt NOTE For SW 32 Ti and SW 32 1 Ti rotors use a lint free cotton swab to apply Spinkote lubri cant 396812 to cap grooves in the bucket tops Match bucket caps with numbered buckets Align the pins on each side of the cap with the guide slots in the bucket Twist the cap clock wise until it stops one quarter turn 7 Attach all buckets loaded or empty to the rotor Loaded buckets must be arranged
73. c tions Then remove it promptly from surfaces While Beckman Coulter has tested these methods and found that they do not damage components no guarantee of decontamination is expressed or implied Consult your laboratory safety officer regarding the proper decontamination methods to use e If the rotor or other components are contaminated with toxic or e a pathogenic materials follow appropriate decontamination proce ey dures as outlined by appropriate laboratory safety guidelines and or other regulations Consult Appendix A to select an agent that will not damage the rotor 1 In U S contact Nuclear Associates New York in Eastern Europe and Commonwealth States contact Victoreen GmbH Munich in South Pacific contact Gammasonics Pty Ltd Australia in Japan contact Toyo Medic Co Ltd Tokyo 2 n U S contact Biodex Medical Systems Shirley New York internationally contact the U S office to find the dealer closest to you Preparative Ultracentrifuge Rotors and Tubes 7 3 Care and Maintenance STERILIZATION AND DISINFECTION 121 C mm C emm INSPECTION Handle O Ring c Al SS Lid O Ring 7 Threads Check for Corrosion oS Overspeed Disk When sterilization or disinfection is a concern consult your labora tory safety officer regarding proper methods to use While Beckman Coulter has tested the following methods and found that they do not dam
74. cate them with Spinkote lubri cant 306812 Failure to keep threads properly lubricated can result in stripped or galled threads and stuck rotor components Rotor plug gaskets a component of vertical tube and near vertical tube rotors do NOT require lubrication but should be checked cleaned and or replaced as required Care and Maintenance 7 6 OVERSPEED DISK REPLACEMENT Centering Tool 331325 The overspeed disk on the rotor bottom is part of the photoelectric overspeed detection system Replace this disk if it is scratched damaged or missing Start with a dry rotor at room temperature the disk will not adhere to a damp surface 1 Pry up the edges of the old disk with a scalpel taking care not to scratch the rotor then peel the disk off 2 Clean the area around the drive hole with acetone to remove any of the old adhesive 3 Insert the centering tool 331325 into the hole 4 Peelthe paper backing off the new disk but do not touch the adhesive Fit it adhesive side down around the centering tool Press the disk firmly to the rotor bottom 5 Remove the tool Allow the disk to set for a minimum of 2 hours TUBE BOTTLE AND ACCESSORY CARE CLEANING Proper care of tubes and bottles involves observing temperature fill volume and run speed limitations as well as careful cleaning and sterilization procedures Do not wash tubes and bottles in a commercial dishwasher detergents and temperat
75. cients the largest particles in the sample traveling to the bottom of the tube first Differential centrifugation is the successive pelleting of particles of decreasing sedimentation velocities using increasingly higher forces and or long run times The relative pelleting efficiency of each rotor is measured by its k factor clearing factor In max min 10 1 o 3600 where w is the angular velocity of the rotor in radians per second 2nRPM 60 or 0 10472 x rpm Fmax is the maximum radius and Tmin 1S the minimum radius After substitution rpm 2 This factor can be used in the following equation to estimate the time t in hours required for pelleting 3 where s is the sedimentation coefficient of the particle of interest in Svedberg units Because s values in seconds are such small numbers they are generally expressed in Svedberg units S where 1 S is equal to 10 13 seconds It is usual practice to use the standard sedimentation coefficient 520 c based on sedimentation in water at 20 C Clearing factors can be calculated at speeds other than maximum rated speed by use of the following formula k P P 2 k ES speed of rotor 4 adj actual run speed Run times can also be calculated from data established in prior exper iments when the k factor of the previous rotor is known For any two rotors a and b t k a a Bi 8 6 th ky where the k factors have been adjusted for the actual run
76. crown LF NL Figure 3 4 Tube Cap Installation The tube must be pushed up past the O ring so that the crown will clamp the tube and NOT the O ring e Thickwall tubes may be filled to within 13 mm of the top but may still collapse if not completely full Stainless steel tubes may be filled to any level Tubes placed opposite each other in the rotor must be filled to the same level FILLING AND CAPPING BOTTLES To prevent spillage and provide support polycarbonate and polypro pylene bottles used in fixed angle rotors must be capped when fill levels exceed the maximum level allowed for uncapped bottles Bottles should be filled to maximum fill levels when spun at maximum rated speeds Unless specified otherwise the minimum recommended volume for bottles is half full this will require reduced rotor speed for optimum labware performance Refer to Table 3 5 and the applicable rotor manual for bottle fill levels and cap requirements 3 22 Preparative Ultracentrifuge Rotors and Tubes Using Tubes Bottles and Accessories Mounting 25 mm 1 in caps clear and black 25 mm 1 in caps red and blue Underside 38 mm 1 1 2 in caps clear 13 mm 1 2 in caps 16 mm 5 s in titanium caps 38 mm 1 1 2 in caps red and blue 16 mm s in all other caps Figure 3 5 Tube Cap Vise Screw the vise to a bench or table top for operation The vise must be correctly mounted with th
77. d with black polyurethane paint Titanium buckets and lids of high performance rotors are usually painted red for identification On some swinging bucket rotors a solid film lubricant coating is added to the bucket flange where the bucket contacts the rotor body The purpose of the coating which is a dull gray in color is to minimize friction and enable the bucket to swing into the rotor bucket pocket more smoothly With use and handling all or part of this coating may wear off this should not affect the rotor performance as the bucket swing up will wear in with use Aluminum rotors are anodized to protect the metal from corrosion The anodized coating is a thin tough layer of aluminum oxide formed electrochemically in the final stages of rotor fabrication A colored dye may be applied over the oxide for rotor identification The O rings or gaskets in fixed angle rotor assemblies or lids and in swinging bucket caps are usually made of Buna N elastomer and maintain atmospheric pressure in the rotor if they are kept clean and lightly coated with silicone vacuum grease Plug gaskets in vertical tube or near vertical tube rotors are made of Hytrel and do not require coating Relatively light rotors have drive pins in the drive hole that mesh with pins on the ultracentrifuge drive hub when the rotor is installed to ensure that the rotor does not slip on the hub during initial accelera tion Heavier rotors do not require the use of drive
78. e clamping knob positioned on the right or crimping of the crown may result iem Cap bottles with three piece cap assemblies as follows oryl Cap 1 Be sure the O ring plug and bottle lip are dry and free of G lubrication Red anodized Noryl Plug Aluminum Cap p 2 Place the O ring on the underside of the plug Neoprene O ring E 3 Insert the plug into the neck of the bottle ensuring that no fluid Ww contacts the O ring N Polycarbonate 4 Tighten the cap by hand Bottle Preparative Ultracentrifuge Rotors and Tubes 3 23 s Using Tubes Bottles and Accessories Table 3 5 Available Bottles Assembly and Operation Bottles are polycarbonate unless otherwise indicated Bottle Required Cap Assembly Volume mL Bottle Maximum Part Dimensions Part and Cap Speed Required Number mm Material Number Assembly Max Min Rotor rpm Adapter 355656 16x 64 Noryl 355604 355615 8 5 8 5 Type 50 50 000 Types 90 Ti 65 000 80 Ti 75 Ti 70 1 Ti 65 355651 16 x 76 Noryl 355604 355603 10 4 5a Type 50 Ti 50 000 Type 40 40 000 11x 79 polypropylene 355672 10 10 Type 28 20 000 342327 870329 Types 70 Ti 60 000 60 Ti 355654 25x89 aluminum 355619 355618 26 3 16 Type 59 2 Ti 55000 Type 50 2 Ti 50 000 Type 42 1 42 000 Noryl 355617 355616 26 3 16 Type 30 30 000 Type 28 20 000 355670 29x 102 polypropylene
79. e of the ot Integrator There are two kinds of experiments in which the integrator is particu larly useful duplicating conditions in a series of rate runs and calculating sedimentation coefficients for rate zonal studies To dupli cate band positions use the integrator to automatically terminate the run at a preselected value of c t In this way even if the set run speed or acceleration is changed for a rotor band positions will be reproduc ible For determining sedimentation coefficients the value of w2t displayed on the integrator at the termination of the run greatly simplifies the arithmetic involved and improves the final result The s t charts for density gradient experiments in swinging bucket rotors provided in publication DS 528 are plots of the relative distance sedimented by a band of particles versus the value of sat They have been calculated for use with 5 to 20 or 10 to 30 wt wt sucrose gradients particle densities of 1 4 and 1 8 g mL and temperatures of 4 and 20 C The following examples illustrate the use of the charts together with the c integrator REPRODUCING BAND POSITIONS refer to Figure B 1 To achieve the best resolution of particle zones the centrifugation duration should be set so that the fastest moving zone of particles will move as close as practical to the bottom of the gradient To determine the centrifugation duration the following must be known an estimate of the sedimentation coefficient o
80. ean mottle Poliomyelitis Polysomes 200 Tobacco mosaic Equine encephalitis Viruses Rous sarcoma 800 Feline leukemia Bacteriophage T2 Microsomes Subcellular Particles Plasma membranes Mitochondria 100 000 Figure 1 3 Sedimentation Coefficients in Svedberg Units for Some Common Biological Materials Preparative Ultracentrifuge Rotors and Tubes 1 9 100 000 95 000 90000 4 85000 4 80 000 75 000 70000 65 000 60 000 1000000 i 900 000 Ti 800000 700 000 i 600000 ce AW SE 500 000 0 3E 400 000 3 160 sli ES dm 300 000 ES 140 1g 40 000 mcs 200 000 T 100 SON 100 000 Jb aE 90 000 T aD 80 000 F 30 000 3d 70000 T w 60 000 Es os 50000 T JL 40 000 pe cm 30 000 S AU 20000 a JL 20 000 wL Ar 10000 aE 9 000 IL ale 8000 il 7000 JE 6000 lL 20 5000 ale 4000 JL L 3000 l 2000 3r 10 1000 10 000 Radial Distance Relative Centrifugal Rotor Speed mm Field x g rpm Figure 1 4 Nomogram Align a straightedge through known values in two columns read the figure where
81. es of Preparative Zonal and Continuous Flow Ultracentrifugation publication DS 468 SW 28 1M and SW 28M rotors no longer manufactured were specially modified versions of the SW 28 1 and SW 28 rotors and are equipped with a mechanical overspeed system These rotors are otherwise identical to the SW 28 1 and SW 28 rotors gt NOTE Although rotor components and accessories made by other manufacturers may fit in the Beckman Coulter rotor you are using their safety in the rotor cannot be ascertained by Beckman Coulter Use of other manufacturers components or accessories in a Beckman Coulter rotor may void the rotor warranty and should be prohibited by your laboratory safety officer Only the components and accessories listed in the applicable rotor manual should be used Preparative Ultracentrifuge Rotors and Tubes 5 3 Using Swinging Bucket Rotors TUBES AND BOTTLES Swinging bucket rotors can accommodate a variety of tube types listed in the applicable rotor manual Refer to Section 3 for tube filling and sealing or capping requirements Observe the maximum rotor speeds and fill volumes listed in the rotor instruction manual ROTOR PREPARATION AND LOADING For runs at other than room temperature refrigerate or warm the rotor beforehand for fast equilibration III NOTE All buckets loaded or empty must be positioned on the rotor body for every run PRERUN SAFETY CHECKS Read all safety information in the rotor ma
82. es that are heat sealed and require no caps polyphenylsulfone used in plugs cap closures cannisters and other accessories Radel is a registered trademark of BP Amoco a method of particle separation based on differential rate of sedimentation using a preformed gradient with the sample layered as a zone on top of the gradient relative centrifugal field the ratio of the centrifugal acceleration at a specified radius and speed rc to the standard acceleration of gravity g according to the following equation RCF ro 8 where r is the radius in millimeters cis the angular velocity in radians per second 27 RPM 60 and g is the standard acceleration of gravity 9807 mm s Thus the relationship between RCF and RPM is RCF 1 12r PM 1000 maximum radius the position of the liquid in the tube at the maximum distance from the axis of rotation when the rotor is at speed minimum radius the position of the liquid in the tube at the minimum distance from the axis of rotation when the rotor is at speed the settling out of particles from a suspension in the earth s field of gravity in the centrifuge this process is accelerated and the particles move away from the axis of rotation sedimentation velocity per unit of centrifugal force nA ll eje SIs 1 ee or a large group of silicone elastomers used in various accessories should be used at temperatures between 59 and 232 C 75 and 450 F Beckm
83. es that will not scratch rotor surfaces 2 Rinse thoroughly with water 3 Air dry the body or buckets upside down Do not use acetone to dry rotors Wipe clean the O rings or gaskets regularly lubricate after cleaning Replace them about twice a year or as required Frequently clean all surfaces that contact O rings Regularly clean the threads of the rotor lid handle buckets cavities and so on with a nonmetal brush and a small amount of concentrated detergent then rinse and dry thoroughly Lubricate the threads as directed under LUBRICATION below Preparative Ultracentrifuge Rotors and Tubes Care and Maintenance DECONTAMINATION Rotors contaminated with radioactive or pathogenic materials must be decontaminated following appropriate laboratory safety guidelines and or other regulations I gt NOTE Strong bases and or high pH solutions can damage aluminum rotors and components e Ifa rotor and or accessories becomes contaminated with radioac tive material it should be decontaminated using a solution that will not damage the anodized surfaces Beckman Coulter has tested a number of solutions and found two that do not harm anodized aluminum RadCon Surface Spray or IsoClean Solution for soaking and Radiacwash III NOTE IsoClean can cause fading of colored anodized surfaces Use it only when necessary and do not soak rotor components longer than the minimum time specified in the IsoClean usage instru
84. f the particle of interest the distance from the axis of rotation it is to travel its density and certain proper ties of the gradient For example to position a protein sample characterized by s of 7 x 10 3 seconds or 7 S and density of 1 4 g mL 37 mm down the length of the centrifuge tube in the SW 60 Ti rotor 37 63 mm or 100 mm from the axis of rotation through a 10 to 30 gradient at 20 C the value of s t must be 0 92 from the figure If the value of swt is divided by s the result is the total centrifugal effect ot 99 B 4 7x10 12 2 o r 131 x 10 rad S The radial distance to the tube meniscus in the SW 60 Ti rotor is 63 mm B 2 Preparative Ultracentrifuge Rotors and Tubes Use of the ot Integrator SW 60 Ti Rotor 120 10 30 Gradient Particle densities are 1 4 and 1 8 g mL 110 100 ae eo pejueuuipes eouejsi jeuonoeJ4 co eo Distance Sedimented in Millimeters 70 0 0 4 0 8 1 2 1 6 2 0 24 2 8 3 2 3 6 4 0 so t SW 60 Ti Rotor 120 5 20 Gradient Particle densities are 1 4 and 1 8 g mL 110 100 ae eo pejueuulpes e uejsi jeuonoeu co eo Distance Sedimented in Millimeters 70 0 0 2 0 4 0 6 0 8 1 0 1 2 1 4 1 6 1 8 2 0 sot Figure B 1 The s t Charts for the SW 60 Ti Rotor Preparative Ultracentrifuge Rotors and Tubes B 3 Use of the ot Integrator This value can be set into the integrator and the integrator used to terminate t
85. f the top of the tube Hill thickwall polypropylene tubes at least half full to maximum level in fixed angle rotors Speed reduction is required Refer to the applicable rotor manual Polyethylene For swinging bucket and fixed angle rotors fill these tubes from half full to maximum level Refer to the applicable rotor manual Preparative Ultracentrifuge Rotors and Tubes 3 13 Using Tubes Bottles and Accessories Stainless Steel CAPPING TUBES TUBE CAP ASSEMBLIES org Setscrew T Nut SS Delrin Washer c Crown m O ring SS Gasket 8 Nylon Insert Stem Tube Because of their strength stainless steel tubes can be centrifuged while filled to any level with all opposing tubes filled to the same level However run speeds must be reduced due to their weight The criteria for speed reduction depends on the tube cap material and the strength of the rotor being used Refer to the applicable rotor manual or Run Speeds for Stainless Steel Tubes publication L5 TB 072 for correct run speeds Caps must be used with thinwall polyallomer and Ultra Clear tubes in fixed angle rotors To prevent spillage thickwall polyallomer poly carbonate and stainless steel tubes must be capped when fill levels exceed the maximum level for uncapped tubes as listed in the applicable rotor manual Cap requirements depend on the tube or bottle material diameter and wall thickness as well
86. f the tube in millimeters L length of the tube in millimeters and tube angle of the rotor being used A Delrin adapter in a rotor cavity or bucket will significantly change the radial distances measured in the tube The equations below can be used to determine r and r min for a given rotor with a Delrin adapter Table 2 3 lists adapter dimensions used in the equations d d d d 1 2 1 2 Paa wel 2 5 sin 10 di d MEER d i 2 e 1 sine 2 cosa 11 2 2 Delrin is a registered trademark of E I Du Pont de Nemours amp Company Preparative Ultracentrifuge Rotors and Tubes Preparative Ultracentrifuge Rotors and Tubes Tubes Bottles and Accessoires where Tmax the distance in millimeters from the axis of rotation to the farthest part of the tube cavity dy outside diameter of the adapter d inside diameter of the adapter L adapter cavity length t thickness of the adapter bottom and 0 tube angle of the rotor being used The values of r nax and r 4 can be used to calculate the k factor and the relative centrifugal field when adapters are used see the equa tions in the Glossary in Appendix E Table 2 3 Dimensions of Delrin Adapters Use these values to calculate radial distances for tubes in Delrin adapters Delrin Adapter Dimensions mm Tube Size Part mL Number d Ap L t 0 8 305527 13 08 5 49 42 09 3 99 356860 18 08 5 36 43 51 3 99 2 30
87. former The seal former should move down the tube stem until it just rests on the tube shoulder Be careful NOT to press the seal former into the tube shoulder it may cause the tube to leak III NOTE Itis very important to apply the heat sink imme diately To do so we recommend that you have it in one hand ready to apply as soon as needed c Remove the Tube Topper IMMEDIATELY place the large end of the heat sink over the seal former Hold it there for a few seconds while the plastic cools do NOT let the seal former pop up If the seal former does pop up the tube may not have an adequate seal and may need to be resealed d Remove the heat sink When the seal former cools remove it by hand or with the removal tool 361668 4 After completing either heat sealing method squeeze the tube gently if the tube contents may be disturbed to test the seal for leaks If the tube does leak try resealing it using Method A 5 Thetube is now ready for centrifugation Seal the remaining tubes 6 Return the Tube Topper to its charging stand when finished Preparative Ultracentrifuge Rotors and Tubes 3 11 Using Tubes Bottles and Accessories FILLING OPEN TOP TUBES OPEN TOP POLYALLOMER TUBES Open top polyallomer tubes are used in swinging bucket and fixed angle rotors Swinging Bucket Rotors Fill all opposing tubes to the same level Thinwall Tubes Fill to within 2 or 3 mm of the top for proper tube wall support
88. get scratched Corrosion begins in scratches and may open fissures in the rotor with continued use The corrosion process accelerates with speed induced stresses The potential for damage from corrosion is greatest in aluminum rotors and components 7 1 Care and Maintenance 7 2 CLEANING Rotor Cleaning Kit 339558 Handle V4 O Ring Wash rotors and rotor components immediately if salts or other corrosive materials are used or if spillage has occurred DO NOT allow corrosive materials to dry on the rotor I gt NOTE Do not wash rotor components or accessories in a dishwasher Do not soak in detergent solution for long periods such as overnight With normal usage wash rotors frequently to prevent corrosion that can begin in scratches N CAUTION Do not immerse or spray a swinging bucket rotor body with water because liquid can become trapped in the hanger mechanism and lead to corrosion 1 Use plastic or wooden tools to remove O rings or gaskets for cleaning do not use metal tools that could scratch anodized surfaces Use a mild detergent such as Beckman Solution 555 339555 diluted 10 to 1 with water and a soft brush to wash rotors and rotor components and accessories Most laboratory detergents are too harsh for aluminum rotors and components The Rotor Cleaning Kit 339558 contains two quarts of Solution 555 and brush
89. gh polycarbonate tubes may be autoclaved doing so greatly reduces the usable life of these tubes Cold sterilization methods are recommended Washing with alkaline detergents can cause failure Crazing the appearance of fine cracks in the tube is the result of stress relaxation and can affect tube performance These cracks will gradually increase in size and depth becoming more visible Tubes should be discarded before cracks become large enough for fluid to escape These tubes have good tolerance to all gradient media except alkalines pH greater than 8 They are satisfactory for some weak acids but are unsatisfactory for all bases alcohol and other organic solvents Polypropylene tubes are translucent and are reusable unless deformed during centrifugation or autoclaving These tubes have good tolerance to gradient media including alkalines They are satisfactory for many Tubes Bottles and Accessories POLYETHYLENE TUBES ULTRA CLEAR TUBES acids bases and alcohols but are marginal to unsatisfactory for most organic solvents They can be used with or without caps in fixed angle rotors Speed reduction is sometimes required with these tubes if run with less than full volume refer to your rotor manual Polyethylene tubes are translucent or transparent and have a good tolerance for use with strong acids and bases They are reusable but cannot be autoclaved In swinging bucket rotors they are used without caps and with or
90. he run when this value is reached Because of deceleration however this value of t will actually be a little too much For a more exact approximation you should make a trial run with an empty rotor and measure the value of t that accumulates during decelera tion from run speed then subtract that value from the total determined from the charts CALCULATING SEDIMENTATION COEFFICIENTS B 4 To calculate sedimentation coefficients the following must be known particle density the distance from the axis of rotation it is to travel specific properties of the gradient run speed and centrifuga tion time The value of w t is used in place of run speed and time For example if a protein of density 1 4 g mL travels 37 mm down the length of the tube in the SW 60 Ti rotor 37 63 mm or 100 mm from the axis of rotation through a 10 to 3096 gradient at 20 C the value of s t is 0 92 from the figure By dividing the value of s t by the product t from the integrator the result is the sedimenta tion coefficient in seconds of the particle s B 5 Preparative Ultracentrifuge Rotors and Tubes Preparative Ultracentrifuge Rotors and Tubes Appendix C The Use of Cesium Chloride Curves This Appendix describes how to determine a maximum rotor speed and the final band positions of particles when performing isopycnic separations using cesium chloride gradients The examples shown here are for the Type 90 Ti rotor only Si
91. he type of rotor the material composition and the rotor s maximum allow able revolutions per minute in thousands referred to as rated speed For example the SW 28 is a swinging bucket rotor with a maximum speed of 28 000 rpm Decimal units that are sometimes part of the rotor name as in the Type 50 2 Ti and the Type 50 4 Ti make it possible to distinguish between different rotors that have the same maximum allowable speed An example of each rotor type is shown in Figure 1 1 Tubes in fixed angle rotors designated Type are held at an angle to the axis of rotation in numbered tube cavities The bodies of some large heavy rotors are fluted to eliminate unnecessary weight and minimize stresses In swinging bucket rotors designated SW containers are held in rotor buckets or attached to the rotor body by hinge pins or a crossbar The buckets swing out to a horizontal position as the rotor acceler ates then seat against the rotor body for support Preparative Ultracentrifuge Rotors and Tubes 1 1 Rotors Fixed Angle Rotor Swinging Bucket Rotor Vertical Tube Rotor Near Vertical Tube Rotor Figure 1 1 Fixed Angle Swinging Bucket Vertical Tube and Near Vertical Tube Rotors In vertical tube rotors designated V tubes are held parallel to the axis of rotation These rotors and the near vertical tube rotors have plugs screwed into the rotor cavities over sealed tubes that restrain the tubes in the cavities and provide
92. iSeal TUBES Preparative Ultracentrifuge Rotors and Tubes OptiSeal tubes are not sealed prior to centrifugation a Noryl plug furnished with each tube is inserted into the stem of filled tubes When the tubes are loaded into the rotor with tube spacers and rotor plugs in vertical tube and near vertical tube rotors in place the g force during centrifugation ensures a tight reliable seal that protects your samples For a detailed discussion on the use of OptiSeal tubes refer to Using OptiSeal Tubes publication IN 189 3 3 Using Tubes Bottles and Accessories Table 3 1 Filling and Capping Requirements for Tubes and Bottles Tube or Bottle Filling Level Requirements Swinging Bucket Rotors Fixed Angle Rotors Vertical and Near Vertical Tube Rotors Polyallomer thinwall tubes thickwall tubes OptiSeal tubes Quick Seal tubes konical Quick Seal tubes konical open top tubes bottles within 2 3 mm of top at least 2 full full and plugged full and heat sealed full and heat sealed within 2 3 mm of top full with cap 1 2 full to max capless level or full with cap Table 3 3 full and plugged full and heat sealed min to max with screw on cap or cap assembly Table 3 3 full and plugged full and heat sealed Ultra Clear open top tubes Quick Seal tubes within 2 3 mm of top full with cap full and heat sealed full and heat sealed Polycarbonate thickwall tube
93. iSeal tubes are being used install a spacer over each plugged tube refer to the applicable rotor manual Leave cavities without tubes completely empty If Quick Seal tubes are being used install spacers and or floating spacers over sealed tubes refer to the applicable rotor manual The particular type of tube support for Quick Seal tubes in fixed angle rotors depends on the length of the tube but the top of the tube must be supported Leave cavities without tubes completely empty 4 Place the lid on the rotor and tighten it as firmly as possible with the handle Screw the handle down clockwise to fully compress the O rings N CAUTION The lid should not touch the tube caps If the lid touches the caps the caps are not seated properly on the tubes Remove the tubes from the rotor and recap them refer to Section 3 Check the tube cavity for foreign matter For runs at other than room temperature refrigerate or warm the rotor beforehand for fast equilibration 4 7 Using Fixed Angle Rotors INSTALLING THE ROTOR Lower the rotor straight down onto the drive hub Carefully lower the rotor straight down onto the drive hub If the rotor has drive pins install it so that the pins are at a 90 degree angle to the pins in the drive hub Careful installation will prevent disturbing the sample or tripping the imbalance detector Refer to the centrifuge instruction manual for detailed operating information
94. ient media Temperature and speed considerations Whether tubes or bottles are to be reused Table 2 1 contains an overview of some of the characteristics of tube and bottle materials A complete list of tubes bottles and accessories is provided in the latest edition of the Beckman Coulter Ultracentrifuge Rotors Tubes amp Accessories catalog BR 8101 available at www beckmancoulter com Preparative Ultracentrifuge Rotors and Tubes 2 1 Tubes Bottles and Accessories Table 2 1 Characteristics and Chemical Resistances of Tube and Bottle Materials Refer to Appendix A for information about specific solutions S F S SM L S 92 o qv ge ke amp D Oo Q gt SE S 4 3 S O Q j S f S S o amp F S 7 amp d o SS ss e o x 7d RY eS Q amp N O gt Q amp e QS c D gt S FS m S VE dS S 8 i oF N o S Gy QU 3 eo ES FS Lg i Oo S S N V e ASA CE QU AUAM E DEA ME ME E Mc DU JE IE IE A Q e x S Sef Se Se Q GSD x O e thinwall transparent yes yes no S U U M S U U U U U S polyallomer thickwall translucent no no yes S S S M S M M U M U S polyallomer Ultra Clear transparent yes yes no S U U S U U U U U polycarbonate transparent no no yes M U U M U U U U U M M polypropylene translucent no no yes S S S M S M S M M transparent polyethylene transparent yes no yes S S S S S S U M M M S translucent cellulose
95. ies Remove the rotor from the centrifuge by lifting it straight up and off the drive hub If a rotor vise is required set the rotor in the rotor vise Remove the rotor plugs taking care to apply downward pressure on the plug adapter to avoid stripping the plugs Remove spacers with the appropriate removal tool or a hemostat Use removal tool 338765 to remove floating spacers Remove tubes with the extraction tool 361668 Refer to Section 3 for sample recovery methods Preparative Ultracentrifuge Rotors and Tubes ROTOR CARE Preparative Ultracentrifuge Rotors and Tubes 7 Care and Maintenance This section provides information on the care of rotors and accessories Included is a list of some common operating problems with suggestions for their solutions Rotors and accessories should be kept in optimal condition to minimize the chance of rotor or labware failure In addition to these instructions observe procedures and precautions provided in individual rotor manuals Appendix A of this manual provides the chemical resistances of rotor and accessory materials to various acids bases salts and solvents Rotor care involves not only careful operating procedures but also careful attention to Regular cleaning decontamination and or sterilization as required Frequent inspection Corrosion prevention and Regular and proper lubrication Do not use sharp tools on a rotor as the surface can
96. ing on wall thickness and are nonwettable although some polyallomer tubes can be chemically treated to make them wettable Polyallomer tubes have good tolerance to all gradient media including alkalines They perform well with most acids many bases many alcohols DMSO and some organic solvents Several types of polyallomer tubes are available Thinwall open top tubes sometimes referred to as straight wall tubes are used in swinging bucket and fixed angle rotors In swinging bucket rotors thinwall tubes should be filled to within 2 or 3 mm of the tube top for proper tube support Caps are usually required in fixed angle rotors Thinwall tubes are designed for one time use and should be discarded after use Thickwall open top tubes offer the convenience of centrifuging partially filled tubes without tube caps in fixed angle and swinging bucket rotors Because the solution reorients during centrifugation the maximum partial fill volume depends on the tube angle For greater fill volumes use tubes with caps Refer to the applicable rotor manual for fill volumes and speed reduction requirements Thickwall tubes are reusable OptiSeal tubes single use tubes designed for use in certain rotors are available in dome top and bell top styles These tubes which come with plastic sealing plugs can be quickly and easily prepared for use without tools or heat Spacers are used to seal the tubes and to support the tops of the tubes during centrif
97. low or stop a rotor by hand e Use only components and accessories that have been designed for use in the rotor being centrifuged refer to the applicable rotor manual The safety of rotor components and accessories made by other manufacturers cannot be ascertained by Beckman Coulter Use of other manufacturers components or accessories in Beckman Coulter prepara tive ultracentrifuge rotors may void the rotor warranty and should be prohibited by your laboratory safety officer The strength of tubes and bottles can vary between lots and will depend on handling and usage Pretest in the rotor using buffer or gradient of equivalent density to the intended sample solution to determine optimal operating conditions Scratches even microscopic ones significantly weaken glass and polycarbonate tubes To help prevent premature failures or hazards by detecting stress corrosion metal fatigue wear or damage to anodized coatings and to instruct laboratory personnel in the proper care of rotors Beckman Coulter offers the Field Rotor Inspection Program FRIP This program involves a visit to your laboratory by a specially trained representative who will inspect all of your rotors for corrosion or damage The representative will recommend repair or replacement of at risk rotors to prevent potential rotor failures Contact your local Beckman Coulter Sales and Service office to request this service It is your responsibility to decontaminate the roto
98. lso for a given rotor speed the maximum CsCl density that can be safely centrifuged at that speed and temperature increases as the fill volume decreases The curves in Figure C 2 show gradient profiles at equilibrium Each curve was generated for the specific rotor speed shown using the maximum CsCl density from Figure C 1 that avoids precipitation at The Use of Cesium Chloride Curves 1 90 1 85 1 80 1 75 1 70 1 65 EM xy 1 60 QV 1 55 uv 1 50 1 45 Wwe We 1 40 Homogeneous CsCl Solution g mL 1 35 NS Sd 1 30 1 25 1 20 TYPE 90 Ti ROTOR 20 C 4C 1 05 1 00 30 Rotor Speed K rpm 70 The dashed line is a repre sentation of the equation RPM 90000 L2g mL p where p density of tube contents and is shown here to illus trate the inability of that equation to guard against CsCl precipitation Figure C 1 Precipitation Curves for the Type 90 Ti Rotor Using combinations of rotor speeds and homoge neous CsCl solution densities that i
99. milar data and examples for other rotors appear in the applicable rotor manual shipped with each rotor Be sure to check the manual for your rotor when calculating run speeds and banding positions Rotor speed controls the slope dp dr of a CsCl equilibrium gradient When planning a separation gradients should be selected so that the density range from the top to the bottom of the gradient is sufficient to encompass the buoyant densities of particles to be separated How ever speeds must often be limited to avoid precipitation of CsCl at the bottom of the gradient The density of crystallized CsCl 4 g mL produces stresses far in excess of the design limits of most rotors Also precipitation will alter the density distribution of the gradient and the position of sample bands The square root reduction formula used to determine maximum rotor speeds when centrifuging dense solutions in plastic tubes does not always guard against CsCl precipitation The square root reduc tion becomes the limiting factor only at relatively high densities and speeds Speed and density combinations that intersect on or below the solid curves in Figure C 1 ensure that CsCl will not precipitate in the Type 90 Ti rotor Curves are provided at two temperatures 20 C black lines and 4 C gray lines Note from Figure C 1 that for a given CsCl density faster rotor speeds can be used as the fill volume in the tube decreases from full to one quarter filled A
100. neral Specifications for Beckman Coulter Preparative Swinging Bucket Rotors Rotors listed in parentheses are no longer manufactured Radial Distances Relative mm k Factors Number of Maximum Centrifugal Tubes x Tube Speed Field x g k g mL g mL g mL Capacity Rotor rpm at max max lev min Factor p 1 3 p 1 5 p 1 7 mL SW 65 Ti 65 000 421 000 89 0 65 1 41 2 46 126 116 112 3x5 SW 60 Ti 60 000 485000 120 3 91 7 63 1 45 126 115 111 6x4 SW 55 Ti 55 000 368000 108 5 84 6 60 8 48 135 123 118 6x5 SW 50 1 50 000 300000 107 3 83 5 59 7 59 165 151 145 6x5 SW 41 Ti 41 000 288 000 153 1 110 2 67 4 124 335 307 295 6 x 13 2 SW 40 Ti 40 000 285000 158 8 112 7 66 7 137 368 338 325 6 x 14 SW 32 Ti 32 000 175000 152 5 109 7 66 8 204 468 428 412 6 x 38 5 SW 32 1 Ti 32000 187 000 162 8 113 6 64 4 228 613 560 536 6x17 SW 30 1 30 000 124000 123 0 99 2 75 3 138 393 360 346 6x8 SW 30 30 000 124000 123 0 99 2 75 3 138 393 360 346 6 x 20 SW 28 1 28 000 150000 171 3 122 1 72 9 276 757 694 668 6x17 SW 28 28 000 141000 161 0 118 2 75 3 246 680 622 600 6 x 38 5 SW 25 1 25 000 90 400 129 2 92 7 562 337 917 840 809 3x34 Maximum speeds are based on a solution density of 1 2 g mL in all swinging bucket rotors Calculated for 5 to 2096 wt wt sucrose at 5 C using the tables in Appendix I of Techniqu
101. ntersect on or below these curves ensures that CsCl will not precipitate during centrifugation Preparative Ultracentrifuge Rotors and Tubes The Use of Cesium Chloride Curves 1 90 1 85 TYPE 90 Ti ROTOR 1 80 Edd i 4C 1 75 ut 1 70 1 65 1 60 1 1 55 1 50 r T D p4 1 45 s E oS 5 S L 140 E L fS 4 1 35 1 30 amp L E f S e L 1 25 is H Fe 1 20 j Figure C 2 CsCl Gradients at Equilibrium Centrifugation of S e homogeneous CsCl solutions at 1 15 amp the maximum allowable speeds e ry from Figure C 1 results in S gradients presented here Lid Density increases from the eS 7 top 34 2 mm to the bottom 10E Ey W s 76 5 mm of the tube f SS e z oo 1 00 34 2 46 1 55 2 63 0 76 5 frin Distance from Axis of Rotation mm fmax Preparative Ultracentrifuge Rotors and Tubes C 3 The Use of Cesium Chloride Curves that speed and temperature The three quarter one half and one quarter filled lines show gradients produced in partially filled tubes Figure C 2 can be used to ap
102. ntrifugation Tubes may become brittle with age and use Dispose of brittle or cracked tubes Thinwall tube volume too low to provide tube wall support Meniscus should be 2 to 3 mm below the tube top Refer to the rotor manual for tube volumes Moisture between the tube and the cavity or bucket can cause the tube to float and collapse Ensure that tubes and tube cavities or buckets are dry before inserting the tubes Reagent used that attacks the tube material Refer to Appendix A for chemical compatibilities of tube material and chemicals Tubes run above their rated speed Refer to the applicable rotor manual for maximum speeds Preparative Ultracentrifuge Rotors and Tubes SYMPTOM Tube Caps Unsure of cap components Setscrew is difficult to remove Setscrew will not seal the tube cap Bottles Bottle leakage bottles with cap assemblies Bottle leakage uncapped bottles Bottle damage Preparative Ultracentrifuge Rotors and Tubes Care and Maintenance POSSIBLE CAUSE AND SUGGESTED ACTION For a complete list of cap components see the Beckman Coulter Ultra centrifuge Rotors Tubes amp Accessories catalog publication BR 8101 available at www beckmancoulter com The hex socket or threads of the screw may be stripped If the screw cannot be removed replace the cap stem Replace the screw and nylon insert if either seems damaged or loose Moisture or lubrication on cap or sealing surface Ensure th
103. nual When using dense solu tions in plastic labware determine maximum run speed using the following square root reduction formula reduced run speed 2 maximum rated speed B 8 where A is the maximum permissible density of the tube contents for a particular rotor from the rotor instruction manual and B is the actual density of the tube contents to be centrifuged When using dense solutions in stainless steel tubes refer to the individual rotor instruction manual or Run Speeds for Stainless Steel Tubes publication L5 TB 072 for allowable speeds e Cesium Chloride Gradients Run speed often must be reduced to avoid the precipitation of CsCl during centrifugation of concentrated CsCl solutions Use the CsCl curves provided in the individual rotor instruction manual to determine run speeds An example of the use of CsCl curves is in Appendix C of this manual e Uncapped Thickwall Plastic Tubes in Fixed Angle Rotors Speed limitations are required to prevent tube collapse when thickwall plastic tubes are centrifuged without the support of tube caps in fixed angle rotors refer to Section 4 e Polycarbonate and Polypropylene Bottles Speed limitations are required to prevent the bottle material from overstressing and deforming refer to Section 2 Adapters When small tubes are used with Delrin adapters run speed often must be reduced due to the increased density of Delrin 1 4 g mL The formula for speed reduction is described
104. nual before using the N rotor 1 Make sure that the rotor body buckets and bucket caps are clean and show no signs of corrosion or cracking qtr P 4 amp 2 Make sure the rotor is equipped with the correct overspeed disk My 60 000 rpm e refer to Section 1 If the disk is missing or damaged replace it as ped described in Section 7 y Pe A ed 3 Check the chemical compatibilities of all materials used Refer to Appendix A 4 Verify that tubes bottles and accessories being used are listed in the appropriate rotor manual 5 4 Preparative Ultracentrifuge Rotors and Tubes Using Swinging Bucket Rotors ROTOR PREPARATION AND LOADING Eca i 4 Gasket Bucket TL a Spacer Tube NF Pug _ Tube X7 g Max Metal Floating Spacer Spacer I Dome Top Bell Top Preparative Ultracentrifuge Rotors and Tubes 1 If the rotor has hinge pins replace any pin that has stripped threads 2 Be sure that bucket threads are clean and lightly but evenly lubricated with Spinkote lubricant 306812 as required 3 Remove the bucket gaskets or O rings and coat them lightly but evenly with silicone vacuum grease 335148 Install gaskets or O rings in the buckets N CAUTION Never run a filled bucket without a gasket or O ring as the bucket contents may be lost leading to rotor imbalance and possible failure 4 Dry the exterior of the tubes Moisture between the tu
105. nwall PA UC 90 Ti 70 Ti 65 60 Ti 55 2 Ti 50 2 Ti 50 Ti 50 45 Ti 42 1 40 35 21 70 Ti 65 60 Ti 55 2 Ti 50 2 Ti 50 Ti 50 45 Ti 42 1 40 35 21 90 Ti 70 Ti 65 60 Ti 55 2 Ti 50 2 Ti 50 Ti 50 45 Ti 42 1 40 35 21 90 Ti 80 Ti 75 Ti 70 1 Ti 90 Ti 80 Ti 75 Ti 70 1 Ti Preparative Ultracentrifuge Rotors and Tubes Continued Using Tubes Bottles and Accessories Table 3 3 Tube Cap Assemblies for Open Top Tubes in Fixed Angle Rotors continued Tube O ring Cap Hex or Assembly Nut Crown Setscrew Insert Gasket Stem Tube Type Rotor Type 25 mm 1 in 302359 301870 302169 338864 302312 301473 302168 thinwall PA 30 UC 3389044 330791 338912f 338864 302312 878188 338908 thickwall PA 30 PC 302133 301870 302169 338864 302312 301473 302138 SS 70 Ti 60 Ti 55 2 Ti 50 2 Ti 42 1 30 331151 330791 331153f 338864 302312 334280 331152 thinwall PA 70 Ti 60 Ti 55 2 Ti UC 50 2 Ti 42 1 3389064 330791 338915f 338864 302312 878188 338908 thickwall PA 70 Ti 60 Ti 55 2 Ti PC 50 2 Ti 42 1 337927 330791 338863 338864 302312 338865 thinwall PA 70 Ti 38 mm 1 1 2 in 326891 301870 326890 808482 302312 346242 326889 thinwall PA 21 UC 3389034 330791 338914f 338864 302312 341767 338909 thickwall PA 21 PC 326905 301870 326890 33886
106. oblem cannot be corrected gt NOTE Use only the labware listed in the applicable rotor manual POSSIBLE CAUSE AND SUGGESTED ACTION Rotor imbalance To balance the rotor load fill all opposing tubes to the same level with liquid of the same density Weight of opposing tubes must be distributed equally Place tubes in a fixed angle near vertical tube or vertical tube rotor symmetrically as illustrated in Section 1 Figure 1 6 Swinging bucket rotor Mishooked bucket loose bucket cap wrong type of bucket mixed bucket types opposing buckets not filled to the same level with liquids of the same density Check loading procedures refer to Section 5 Rotor vise not used wrong tool used incorrect torque or insufficient pressure on plug adapter when tightening rotor plugs Observe careful tightening procedures Threads contaminated with dirt dried lubricant or metal particles or threads insufficiently lubricated cause rotor components to stick Do not use excessive force to loosen components Contact your Beckman Coulter representative Routinely clean metal threads with concentrated Solution 555 339555 then lubricate them with Spinkote 306812 Not an operational problem Care and Maintenance SYMPTOM Tubes Tube leakage Tubes with cap assemblies Tubes with snap on caps Uncapped tubes OptiSeal tubes Quick Seal tubes Tube cracking Tube collapse POSSIBLE CAUSE AND SUGGESTED ACTION
107. of rotation Glossary of Terms E 2 clearing factor k clearing time t CsCl CsSO Delrin density density separation differential separation EPDM ethidium bromide fixed angle rotor g Max HDPE calculated for all Beckman Coulter ultracentrifuge rotors as a measure of the rotor s relative pelleting efficiency _ In Tmax min 10 i i 3600 or jiu 55209 OI Uma RPM 1000 t k s where t is time in hours k is the clearing factor of the rotor and s is the sedimentation coefficient in Svedberg units S cesium chloride a high density salt used in solution in isopycnic separations to separate particles based on their density cesium sulfate a salt similar to CsCl that will form its own gradient in solution thermoplastic material acetal homopolymer used for most tube adapters Delrin is a registered trademark of E I Du Pont de Nemours amp Company mass per unit volume a centrifugal separation process based on differences in particle densities a centrifugal separation process based on differences in particle sizes ethylene proplyene rubber used for O rings and pad adapters should be used at temperatures between 57 and 120 C 70 and 250 F a fluorescent intercalating orange dye used commonly in the separation of DNA and in gel electrophoresis a rotor in which the tubes are held at an angle usually 20 to 45 degrees from the axis of rotation a system of cen
108. oly ethylene Noryl or cellulose propionate can be autoclaved at 121 C for about 20 minutes Note that autoclaving reduces the lifetime of polycarbonate tubes Also polyallomer tubes may be permanently deformed if they are autoclaved many times or if they are handled or compressed before they cool Tubes and bottles should be placed open end down or supported in a rack if autoclaved Do not autoclave plastic adapters or spacers N CAUTION Do not autoclave tubes or bottles with caps on Pressure in a sealed container can cause an explosion Pressures within the autoclave can cause partially sealed containers to collapse when the autoclave vents 7 7 Care and Maintenance Table 7 1 Tube and Bottle Sterilization and Disinfection This information is provided as a guide to the use of sterilization and disinfection techniques for tube materials Cold sterilization results shown are for short duration 10 minute soak periods reactions may differ with extended contact Refer to Appendix A of this manual for information about specific solutions Sodium Hydrogen Glutaral Tube Bottle Autoclave UV Ethylene Formal Ethanol Hypochlorite Peroxide dehyde Phenolic Material 121 C Irradiation Oxide dehyde 70 10 10 2 Derivatives polyallomer yes no yes yes yes yes yes yes no Ultra Clear no no yes yes yes yes yes yes no polycarbonate yes4 no yes yes3 no yes yes yes no polypropylene yes no yes
109. on as a function of sedimentation coefficient Swinging bucket rotors are best applied for rate zonal studies in which maximum resolution of sample zones are needed or pelleting runs where it is desirable for the pellet to be in the exact center of the tube bottom Gradients of all shapes and steepness can be used Refer to Section 5 for specific information about the use of swinging bucket rotors Vertical tube rotors hold tubes parallel to the axis of rotation therefore bands separate across the diameter of the tube rather than down the length of the tube see Figure 1 2 Vertical tube rotors are useful for isopycnic and in some cases rate zonal sepa rations when run time reduction is important Only Quick Seal and OptiSeal tubes are used in vertical tube rotors making tube caps unnecessary Refer to Section 6 for specific information about the use of vertical tube rotors M Near vertical tube rotors are designed for gradient centrifugation when there are components in a sample mixture that do not partici pate in the gradient The reduced tube angle of these rotors significantly reduces run times from the more conventional fixed angle rotors while allowing components that do not band under separation conditions to either pellet to the bottom or float to the top of the tube Like the vertical tube rotors near vertical tube rotors use only Quick Seal and Opti
110. ons is a discussion of rotor materials components and centrifu gation techniques Section 2 describes various tubes bottles adapters and spacers to help you choose a particular tube or bottle for your application Section 3 provides instructions for using tubes or bottles and related accessories Section 4 contains step by step procedures for preparing a fixed angle rotor for a centrifuge run Similar information for swinging bucket rotors is in Section 5 and Section 6 contains the same type of informa tion for vertical tube and near vertical tube rotors Analytical continuous flow and zonal rotors are not covered in this manual e Section 7 provides rotor tube bottle and accessory care and maintenance information as well as some diagnostic hints Please read it Good rotor care results in longer rotor life e Several appendixes contain information that may be of special interest Appendix A lists chemical resistances for rotor and accessory materials to help determine compatibility with a variety of solutions Appendix B contains information about the use of the c t integrator Appendix C describes the use of cesium chloride curves Appendix D contains reference information on some commonly used gradient materials Appendix E contains a glossary of terms used in this manual Appendix F lists references for further reading Preparative Ultracentrifuge Rotors and Tubes SECTION 1 SECTION
111. ottles i2 aso ees acte ee Cera qae d eee waka 2 7 Temperature Limits llle 2 8 Spacers and Floating Spacers 0 0 cece ee eee eee 2 8 Adapters zx ce rem stands iR eta dents say donde wala ba ees 2 10 USING TUBES BOTTLES AND ACCESSORIES Gradient Preparation 0 0 eee eee eee 3 1 Cesium Chloride Gradients 00 eee ee ee eee eee 3 2 General Filling and Sealing or Capping Requirements 3 3 Filling and Plugging OptiSeal Tubes 0 004 3 3 Filling the Tubes 0 0 0 eee eee eee 3 5 Seating the Tube Plugs 0 0 0 0 c eee eee eee 3 7 Filling and Sealing Quick Seal Tubes 00 0 3 8 Method A With the Seal Guide 00 4 3 9 Method B Without the Seal Guide 3 11 Filling Open Top Tubes 0 0 0 0 eee eee eee eee 3 12 Open Top Polyallomer Tubes 0 000 000 000 3 12 Other Open Top Tubes 00 0 0 eee 3 12 Capping Tubes i260 erorii erei en Ro WR ee ee 3 14 Tube Cap Assemblies 2 0 0 eee 3 14 Inspecting and Lubricating Tube Caps 3 19 Assembling Tube Caps 2 0 0 0 cece ee eee eee 3 19 Filling and Capping Bottles 3 22 Preparative Ultracentrifuge Rotors and Tubes SECTION 4 SECTION 5 Page Sample Recovery lsseeelee eee eee 3 25 Capped Tubes 0 cece eee ee eee 3 25 OptiSeall Tubes cis edet em Rete eat 3 2
112. pins For swinging bucket rotors an indentation on the rotor adapter or the position of the mechanical overspeed cartridges see Overspeed Protection below indicates the location of the drive pins In this way the pins can be properly aligned without lifting the rotor and dislocating the buckets Rotors ROTOR SELECTION Selection of a rotor depends on a variety of conditions such as sample volume number of sample components to be separated particle size desired run time desired quality of separation type of separation and the centrifuge in use Fixed angle swinging bucket vertical tube and near vertical tube rotors are designed to provide optimal separations for a variety of sample types For especially large sample volumes continuous flow and zonal rotors are available Fixed angle rotors are general purpose rotors that are especially a useful for pelleting subcellular particles and in short column banding of viruses and subcellular organelles Tubes are held at an angle usually 20 to 45 degrees to the axis of rotation in numbered tube cavities The tube angle shortens the particle pathlength see Figure 1 2 compared to swinging bucket rotors resulting Nl in reduced run times Refer to Section 4 for specific information about the use of fixed angle rotors Swinging bucket rotors are used for pelleting isopycnic studies separation as a function of density and rate zonal studies sepa rati
113. proximate banding positions of sample particles In general lower speeds generate gradients with shallow slopes bands will be farther apart Higher speeds generate gradients with steep slopes where bands will be closer together Gradient curves not shown can be interpolated III NOTE The curves in Figures C 1 and C 2 are for solu tions of CsCl salt only If other salts are present in significant concentrations the overall CsCl concentration or the rotor speed must be reduced For example a quarter filled tube of a 1 52 g mL homogeneous CsCI solution at 20 C may be centrifuged at 80 000 rpm see Figure C 1 The segment of the 80 000 rpm curve Figure C 2 from the quarter filled line to 1 86 g mL at the tube bottom represents this gradient The same solution in a half filled tube Figure C 1 may be centri fuged no faster than 68 000 rpm Using Figure C 2 interpolate between the 60 000 rpm and 70 000 rpm curves and draw the new 68 000 rpm gradient curve to the half filled level The same solution in a three quarter filled tube may be centrifuged at 59 000 rpm Figure C 2 shows the gradient profile use the three quarter filled segment only A tube full of the 1 52 g mL CsCl solution may be centrifuged no faster than 53 000 rpm interpolate and draw in the new gradient profile TYPICAL EXAMPLES FOR DETERMINING CsCl RUN PARAMETERS Example A A separation that is done frequently is the banding of plasmid DNA in cesium chloride wi
114. r For example two three four or six tubes can be arranged symmetrically in a six place rotor N CAUTION For swinging bucket rotors attach ALL buckets whether loaded or empty For vertical tube and near vertical tube rotors insert spacers and rotor plugs ONLY in holes containing loaded tubes If sample quantity is limited and the rotor is not balanced do one of the following to balance the rotor depending on the rotor in use e Load the opposite rotor cavities or buckets with tubes containing a liquid of the same density as opposing tubes e Use smaller tubes with adapters or smaller Quick Seal tubes with floating spacers to distribute the sample symmetrically Use thickwall tubes partially filled to distribute sample to additional tubes Layer alow density immiscible liquid such as mineral oil on top of the sample to fill opposing tubes to the same level Do not use an oil overlay in Ultra Clear tubes Rotors are specifically designed to withstand a maximum load that is volume and density of the rotor contents at maximum rated speed At greater speeds or at rated speeds with heavier loads rotors are subject to failure It is the operator s responsibility to limit rotor speed when centrifuging dense solutions or when using heavy tubes refer to ALLOWABLE RUN SPEEDS below Preparative Ultracentrifuge Rotors and Tubes ote iy 75 000 rpm p 24 Sector a 334217 a Pant Drive Pin Cartridge Cart
115. r tripping the imbalance detector N CAUTION If hook on buckets have been jarred during installation check them with a mirror for proper vertical positioning see Figure 5 2 Remove the rotor to correct any unhooked buckets 3 Refer to the centrifuge instruction manual for detailed operating information Figure 5 2 Checking Hook on Bucket Positions After the Rotor is Installed Note the partially unhooked bucket on the right 5 7 Using Swinging Bucket Rotors REMOVAL AND SAMPLE RECOVERY N CAUTION If disassembly reveals evidence of leakage you should assume that some fluid escaped the rotor Apply appropriate decontamination procedures to the centrifuge and accessories 1 Remove the rotor from the centrifuge by lifting it straight up and off the drive hub 2 Set the rotor on the rotor stand and carefully remove the buckets lift buckets off crossbars or unscrew the hinge pins 3 Remove the bucket caps and use the appropriate removal tool to remove the spacers and tubes 4 Remove adapters using the appropriate removal tool While pressing the rubber tip sede uat Extractor NOTE pull the tool EFA If conical shaped adapters that support konical Bar tubes are difficult to remove after centrifugation of the cavity an extractor tool 354468 is available to facili tate removal 5 Refer to Section 3 for sample recovery methods 5 8 Preparative Ultracentrifuge Rotors and Tubes D
116. r emphasize the need for aerosol protection Handle other infectious samples according to good laboratory procedures and methods to prevent spread of disease Because spills may generate aerosols observe proper safety precautions for aerosol containment Do not run toxic pathogenic or radioactive materials in a rotor without taking appropriate safety precautions Biosafe containment should be used when Risk Group II materials as identified in the World Health Organi zation Laboratory Biosafety Manual are handled materials of a higher group require more than one level of protection Dispose of all waste solutions according to appropriate environmental health and safety guidelines e If disassembly reveals evidence of leakage you should assume that some fluid escaped the rotor Apply appropriate decontamination procedures to the ultracentrifuge and accessories Preparative Ultracentrifuge Rotors and Tubes Mechanical Safety Use only rotors and accessories designed for use in the ultracentrifuge you are operating Do not use rotors in ultracentrifuges with any classification except those indicated in the rotor manual or engraved on the rotor Rotors are designed for use at the rated speeds indicated however speed reductions may be required because of weight considerations of tubes adapters or the density of the solution being centrifuged Be sure to observe the instructions in the appropriate rotor manual NEVER attempt to s
117. rack Extraction Tool 2 Press down on the rack insert on each side of the tube being Rack Insert unplugged to hold the tube in place during plug removal x gt NOTE Do not hold onto or squeeze the tubes Tube contents will splash out when the plug is removed if pressure is applied to the tube 3 While pressing down on the rack insert use the extraction tool to firmly grasp the plug 4 Useaslight twisting motion to slowly release any residual internal pressure when pulling the plug assembly from the tube 5 Repeat for each tube QUICK SEAL TUBES There are several methods of recovering fractions from Quick Seal tubes One of the following procedures may be used I gt NOTE m om If you plan to collect particles from the tube side provide an or bottom first create an air passage by snipping air inlet the stem or inserting a hollow hypodermic needle in the top of the tube e Puncture the side of the tube just below the band with a needle and i syringe and draw the sample off Take care when piercing the tube H to avoid pushing the needle out the opposite side 3 28 Preparative Ultracentrifuge Rotors and Tubes Ns Sample out p CentriTube Slicer 347960 Using Tubes Bottles and Accessories Puncture the bottom of the tube and collect the drops e Aspirate the sample from the tube top by snipping off the tube stem then aspirating the sample with a Pasteur pipette or needle
118. rdless of the type of separation gt NOTE If you plan to collect particles from the tube side or bottom first create an air passage by removing the tube plug see instructions below or inserting a hollow hypodermic needle in the top of the tube 3 26 Preparative Ultracentrifuge Rotors and Tubes Using Tubes Bottles and Accessories 4 Use one of the following methods to retrieve the sample SN Sample out CentriTube Slicer iu 347960 Preparative Ultracentrifuge Rotors and Tubes Puncture the side of the tube just below the sample band with a needle and syringe and draw the sample off Take care when piercing the tube to avoid pushing the needle out the opposite side Puncture the bottom of the tube and collect the drops Aspirate the sample from the tube top by removing the tube plug see instructions below then aspirating the sample with a Pasteur pipette or needle and syringe Slice the tube using the Beckman CentriTube Slicer 303811 Refer to publication L TB 010 for instructions for using the CentriTube Slicer Use CentriTube Slicer 347960 and Centri Tube Slicer Adapter 354526 for 13 mm tubes Tubes are pressurized after centrifugation so pierce the tube top with a needle to relieve pressure before slicing Using Tubes Bottles and Accessories Removing Plugs from Tubes Place the tube rack insert over the tubes in the
119. ridge Preparative Ultracentrifuge Rotors and Tubes Rotors Rotors are protected from exceeding their maximum rated speed to help prevent failure and damage to the rotor and the instrument Two overspeed protection systems are used in Beckman Coulter preparative ultracentrifuges e Optima L and LE classified R and Optima XL and L XP classi fied S as well as Models L2 65B L2 75B and L3 classified G Models L5 L5B L8 and L8M classified H and Model L7 classified R have a photoelectric overspeed system This system includes a photoelectric device in the rotor chamber next to the drive hub and an overspeed disk on the rotor bottom Earlier model ultracentrifuges classified other than G H R or S and some F have a mechanical overspeed system All Beckman Coulter preparative rotors are shipped with an over speed disk attached and are therefore protected from overspeeding in instruments with the photoelectric system These instruments will not operate unless an overspeed disk is attached to the installed rotor The disk has alternating sectors of reflecting and nonreflecting material The number of sectors on the disk is a function of the rotor s maximum allowable speed During centrifugation if the reflective segments pass over the photoelectric pickup faster than the indicated set speed the drive will automatically decelerate to the allowed speed The earlier model ultracentrifuges classified A B C D N
120. rifuge Datasheet Optima M L 100 XP Ultracentrifuge Datasheet OptiSeal Ultracentrifuge Tubes Rapid Isolation of Both RNA amp DNA from Cultured Cell Purity Antigenicity and Immunogenicity ULTRACENTRIFUGE ROTOR WARRANTY All Beckman Coulter ultracentrifuge Fixed Angle Vertical Tube Near Vertical Tube Swinging Bucket and Airfuge rotors are warranted against defects in materials or workmanship for the time periods indicated below subject to the Warranty Conditions stated below Preparative Ultracentrifuge Rotors 5 years No Proration Analytical Ultracentrifuge Rotors 5 years No Proration ML and TL Series Ultracentrifuge ROLES ace edente e epe 5 years No Proration Airfuge Ultracentrifuge Rotors 1 year No Proration For Zonal Continuous Flow Component Test and Rock Core ultracentrifuge rotors see separate warranty Warranty Conditions as applicable 1 This warranty is valid for the time periods indicated above from the date of shipment to the original Buyer by Beckman Coulter or an authorized Beckman Coulter representative 2 This warranty extends only to the original Buyer and may not be assigned or extended to a third person without written consent of Beckman Coulter 3 This warranty covers the Beckman Coulter Centrifuge Systems only including but not limited to the centrifuge rotor and accessories and Beckman Coulter shall not be liable for damage to or loss of the u
121. rifuged when filled to any level Because of their weight however run speeds must often be reduced see publication L5 TB 072 Stainless steel tubes can be used indefinitely if they are undamaged and not allowed to corrode They may be autoclaved after use as long as they are thoroughly dried before storage kKONICAL TUBES konical tubes used with conical adapters in swinging bucket rotors to optimize pelleting separations have a conical tip that concentrates the pellet in the narrow end of the tube The narrow bottom also reduces the tube s nominal volume and minimizes the amount of gradient material needed when pelleting through a dense cushion They are available in polyallomer and Ultra Clear The konical tubes come in both open top and Quick Seal tube designs The Quick Seal type have bell shaped tops to fit the floating spacers in the g Max system for smaller volume runs with faster pelleting BOTTLES Bottles are available in polycarbonate hard and clear polypropylene translucent and polyallomer translucent Threaded top polycarbonate bottles are available for many fixed angle rotors They have a liquid tight cap assembly and are easy to use Caps and plugs if applicable should always be removed before autoclaving C Type 16 and Type 28 rotors no longer manufactured use capped polypropylene bottles in addition to polycarbonate bottles The Type 19 rotor uses a polyallomer bottle with a
122. rotor cavities A tube will sometimes come out of the rotor cavity along with the spacer Separate the tube from the spacer with a twisting motion Spacer Removal Tool III NOTE SW 32 Ti and SW 28 rotors only Use the spacer removal tool 338765 to remove the spacer and tube together from the rotor bucket Place the tubes in the rack Grasp the tube and use the spacer removal tool in a lifting and twisting motion to remove the spacer gt NOTE Centrifugation causes a slight vacuum to build up in the tube cavity occasionally resulting in a suction effect when removing the tubes from the rotor This effect is especially pronounced in a rotor that has been centrifuged at a low temperature A brief delay approximately 5 minutes after the rotor comes to rest before removing the tubes will make tube removal easier If you experience difficulties in removing the tubes from the rotor use a gentle twisting or rocking motion and remove the tube slowly to avoid sample mixing 2 Remove the tube with the extraction tool 361668 grasping the base of the stem only do NOT try to remove the tubes by pulling on the plugs Some tube deformation occurs during centrifugation which causes a slight internal pressure to develop inside the tube Extraction Tool 3 Place the tubes back into the tube rack Openings in the rack allow EM the tubes to be pierced either from the bottom or sides permitting fractions to be easily collected rega
123. rs and accessories before requesting service by Beckman Coulter Field Service Preparative Ultracentrifuge Rotors and Tubes LR IM 24 December 2007 C COULTER ROTORS AND TUBES For Beckman Coulter Preparative Ultracentrifuges User s Manual 2007 Beckman Coulter Inc Produced in the U S A CA 1207 PDF SCOPE OF THIS MANUAL This manual contains general information for properly preparing a rotor for centrifugation in a Beckman Coulter preparative ultracentrifuge This manual should be used with the individual rotor instruction manual packed with each rotor The rotor manuals provide specific information for each rotor including special operating procedures and precautions tube bottle and adapter part numbers and equations to calculate maximum allowable rotor speeds Each manual has a code number in the upper right hand corner of the cover page that can be used for reordering To reorder contact customer service at 1 800 742 2345 in the United States outside the U S contact your local Beckman Coulter representative A lot of information is compiled in this manual and we urge you to read it carefully especially if this is your first experience with Beckman Coulter products n Section 1 you will find descriptions by usage of Beckman Coulter s currently produced preparative ultracentrifuge rotors this should help you determine the appropriate rotor to use for a particular application Also included in this secti
124. rsion for Cesium and Rubidium Salts at 20 C D 5 Contents Xi Classification Program All Beckman Coulter preparative ultracentrifuges are classified according to the size and protective barrier of the rotor chamber the type of overspeed detection system and the degree of updating the instruments have if any Preparative ultracentrifuges should have a decal above the rotor chamber opening on top of the instrument or on the chamber door indicating their classification letter Beckman Coulter rotors are then specified for use in particular instrument classes In June 1954 a major reclassification program was established to ensure continued safety to users of older ultracentrifuges and or rotors This reclassification of instruments and rotors is outlined below It is essen tial that you use this program to determine which rotors may be safely run in which instruments Rotors in parentheses are no longer manufactured A CAUTION Rotors without mechanical overspeed devices should not be used in ulracentrifuges classified other than G H R or S INSTRUMENT CLASSIFICATION ROTORS THAT MAY BE USED IN THIS INSTRUMENT All Model L s classified A Type 40 Type 40 2 Type 40 3 SW 50 1 SW 25 1 and A1 15 All Model L s classified B Type 50 Ti Type 50 3 Ti Type 50 Type 40 Type 40 2 Type 40 3 SW 50 1 SW 30 SW 30 1 SW 25 1 and zonals All Model L2 50 s classified C
125. s thickwall bottles at least 2 full 1 2 full to max capless level or full with cap or cap assembly Table 3 3 min to max with screw on cap or cap assembly Table 3 3 Stainless Steel tubes any level any level with cap or cap assembly Table 3 3 Cellulose Propionate tubes full 1 2 to max capless level no cap Polypropylene tubes and bottles at least 1 2 full 1 2 to max capless level or full with cap or cap assembly Polyethylene tubes at least 1 2 full 1 2 to max capless level or full with cap Corex Pyrex tubes and bottles at least 1 2 full 1 2 to max capless Preparative Ultracentrifuge Rotors and Tubes Using Tubes Bottles and Accessories FILLING THE TUBES For filling convenience use the appropriate eight tube rack listed in Table 3 2 1 Use a pipette or syringe to fill each tube leaving no fluid in the stem see Figure 3 1 Overfilling the tube can cause overflow when the plug is inserted however too much air can cause the tube to deform and disrupt gradients and sample bands as well as increasing the force required to remove the tube from the cavity after centrifugation gt NOTE If air bubbles occur in the tube shoulder area tilt and rotate the tube before it is completely filled to wet the tube Homogeneous solutions of gradients and sample may be loaded into the tubes and centrifuged immediately See GRADIENT PREPARATION above
126. s in centimeters and h is the interband separa tion in centimeters Preparative Ultracentrifuge Rotors and Tubes Preparative Ultracentrifuge Rotors and Tubes Appendix D Gradient Materials This Appendix contains reference information on commonly used gradient materials General instructions for filling and sealing tubes including gradient preparation are contained in Section 3 Gradient material selection depends on a number of factors including the type of separation to be performed Sucrose is used for rate zonal and isopycnic separations and cesium chloride is often used for isopycnic separations The basic requirement is that the gradient permit the type of separation Additional considerations in selecting a gradient material include the following ts density range should be sufficient to permit separation of the particles of interest by the chosen density gradient technique without overstressing the rotor t should not affect the biological activity of the sample tshould be neither hyperosmotic or hypoosmotic when the sample is composed of sensitive organelles t should not interfere with the assay technique e It should be removable from the purified product e t should not absorb in the ultraviolet or visible range e t should be inexpensive and readily available more expensive materials should be recoverable for reuse t should be sterilizable t should not be corrosive to the rotor t
127. sary turn the tube slightly to break any vacuum seal created between the tube and the cavity and pull the tube out Use the hex key end of the removal tool to remove the cap setscrew but try not to squeeze the tube With the setscrew removed supernatant liquid can be withdrawn from the tube or the tube bottom can be punctured for fraction collection Extract capless tubes using forceps or a hemostat and OptiSeal or Quick Seal tubes with the removal tool 361668 To remove polycarbonate bottles with black Noryl caps insert the crossbar end of the removal tool 335381 into the cap slot and turn until the crossbar is past the slot Pull the bottle out For bottles with red aluminum caps depress the button of the removal tool 878133 and insert the end of the tool into the cap hole Release the button and pull the bottle out 5 Remove adapters using the appropriate removal tool 6 Refer to Section 3 for sample recovery methods 5 Using Swinging Bucket Rotors This section contains instructions for using swinging bucket rotors in preparative ultracentrifuges In addition to these instructions observe procedures and precautions provided in the applicable rotor and ultracentrifuge manuals Refer to Section 2 for labware selection information and Section 3 for recommended filling and sealing or capping requirements and for sample recovery procedures Refer to Section 7 for information on the care of rotors and acce
128. sedimentation coefficient and density to the bottom of a 5 to 20 gradient 0 where s is the sedimentation coefficient in Svedberg units S A more accurate way to estimate run times in rate zonal studies is to use the sc t charts available in Use of the wt Integrator publication DS 528 If the values of s and w are known and gradients are either 5 to 2096 or 10 to 3096 wt wt sucrose you can use the charts to calculate the run time t Conversely if the value of 2t is known sedimentation coefficients can be estimated from zone positions Refer to Appendix B of this manual for an explanation of the s charts In most cases when banding two or three components by rate zonal separation run times can be considerably reduced by using reduced fill levels Tubes are partially filled with gradient but the sample volume is not changed however gradient capacity will be reduced Thickwall tubes should be used when this technique is employed since thinwall tubes will collapse if not full If swinging bucket rotors are used with preformed shallow gradients 5 to 20 or if fixed angle vertical tube or near vertical tube Preparative Ultracentrifuge Rotors and Tubes Rotors rotors are used with any preformed gradient use the slow acceleration control on your ultracentrifuge Slow acceleration will protect the sample to gradient interface and slow deceleration will maintain the integrity of the separation during the reorient
129. ser s sample non Beckman Coulter tubes adapters or other rotor contents 4 This warranty is void if the Beckman Coulter Centrifuge Sys tem is determined by Beckman Coulter to have been operated or maintained in a manner contrary to the instructions in the operator s manual s for the Beckman Coulter Centrifuge System components in use This includes but is not limited to operator misuse abuse or negligence regarding indicated maintenance procedures centrifuge and rotor classification requirements proper speed reduction for the high density of certain fluids tubes and tube caps speed reduction for precipi tating gradient materials and speed reduction for high tempera ture operation 5 Rotor bucket sets purchased concurrently with or subsequent to the purchase of a Swinging Bucket Rotor are warranted only for a term co extensive with that of the rotor for which the bucket sets are purchased 6 This warranty does not cover the failure of a Beckman Coulter rotor in a centrifuge not of Beckman Coulter manufacture or if the rotor is used in a Beckman Coulter centrifuge that has been modified without the written permission of Beckman Coulter or is used with carriers buckets belts or other devices not of Beckman Coulter manufacture 7 Rotor parts subject to wear including but not limited to rotor O rings VTi NVT TLV MLN and TLN rotor tube cavity plugs and gaskets tubing tools optical overspeed disks bear ings
130. should not be flammable or toxic to the extent that its aerosols could be hazardous The following charts are provided as a reference for information on commonly used gradient materials Gradient Materials Table D 1 Commonly Used Gradient Materials with Their Solvents Maximum Density Materials Solvent at 20 C Sucrose 66 H20 1 32 Sucrose 65 D20 1 37 Silica sols H2O 1 30 Diodon H20 1 37 Glycerol H20 1 26 Cesium chloride H20 1 91 D20 1 98 Cesium formate H20 2 10 Cesium acetate H20 2 00 Rubidium chloride H20 1 49 Rubidium formate H20 1 85 Rubidium bromide H2O 1 63 Potassium acetate H20 1 41 Potassium formate H20 1 57 D20 1 63 Sodium formate H20 1 32 D20 1 40 Lithium bromide H20 1 83 Lithium chloride D20 1 33 Albumin H20 1 35 Sorbitol H20 1 39 Ficoll H2O 1 17 Metrizamide H20 1 46 Preparative Ultracentrifuge Rotors and Tubes Gradient Materials Table D 2 Density Refractive Index and Concentration Data Cesium Chloride at 25 C Molecular Weight 168 37 Density Refractive 96 by mg mL of Density Refractive 96 by mg mL of g cm3 Index nD Weight Solution Molarity g cm3 Index nD Weight Solution Molarity 1 0047 1 3333 1 10 0 0 056 1 336 1 3657 34 454 2 2 698 1 0125 1 3340 2 20 2 0 119 1 3496 1 3670 35 472 4 2 806 1 0204 1 3348 3 30 6 0 182 1 363 1 3683 36 490 7 2 914 1 0284 1 3356 4 41 1 0 244 1 377 1 3696 37 509 5 3 026 1 0365 1 3364 5 5
131. speed used 1 s dr dt x 1 w r where dr dt is the sedimentation velocity 1 8 Preparative Ultracentrifuge Rotors and Tubes Rotors Figure 1 3 lists sedimentation coefficients for some common biologi cal materials The k factors at maximum speeds for Beckman Coulter preparative rotors are provided in the table of general specifications in each rotor use section The centrifugal force exerted at a given radius in a rotor is a function of the rotor speed The nomogram in Figure 1 4 allows you to determine relative centrifugal field RCF for a given radius and rotor speed Run times can be shortened in some rotors by using the g Max system The short pathlength means less distance for particles to travel in the portion of the tube experiencing greatest centrifugal force and hence shortened run times Run times can also be short ened in some rotors by using partially filled thickwall polyallomer Cytochrome c Collagen Yeast tRNA Albumin EI Soluble Proteins Luteinizing hormone Immunoglobulin G Aldolase Nucleic Acids Catalase a Macroglobulin E coli rRNA Calf liver DNA 20 Ribosomal subunits 40 Vesicular stomatitis virus RNA Bacteriophage T5 DNA 60 Bacteriophage T2 amp T4 DNAs Ribosomes Broad b
132. ssories DESCRIPTION Swinging bucket rotors see Figure 5 1 are most frequently used for density gradient separations either isopycnic or rate zonal Refer to Table 5 1 for general rotor specifications Tubes in swinging bucket rotors are held in the rotor buckets Buckets are attached to the rotor body by hinge pins or a crossbar The buckets swing out to a horizontal position as the rotor accelerates then seat against the rotor body for support Bucket and rotor body positions are numbered for operator convenience Each bucket is sealed by an O ring or gasket between the bucket and the bucket cap Caps are either a small flat cap tightened with a screwdriver or a cap that is integral with the hanger mechanism screwed into the bucket by hand Some swinging bucket rotors have a hollow handle on top designed for use with a temperature sensing thermistor and a rotor stabilizer features of the early model ultracentrifuges Models L and L2 Operators using Model L2 ultracentrifuges should refer to individual rotor manuals for the stabilizer level to be used for Beckman Coulter s newer rotors Preparative Ultracentrifuge Rotors and Tubes 5 Axis of Rotation Axis of Rotation Axis of Rotation Axis of Rotation min SW 60 Ti j SW 40 Ti T SW 28 SW 28 1 Figure 5 1 Swinging Bucket Rotors Using Swinging Bucket Rotors Table 5 1 Ge
133. stributed its density range will encompass the density of the particle s of interest Refer to Appendix C for an explanation of the use of the CsCl curves Preparative Ultracentrifuge Rotors and Tubes Using Tubes Bottles and Accessories GENERAL FILLING AND SEALING OR CAPPING REQUIREMENTS See Table 3 1 for general filling and sealing or capping requirements for tubes and bottles used in preparative rotors Maximum fill volume includes sample and gradient Refer to individual rotor manuals for specific filling and capping requirements N WARNING Handle body fluids with care because they can transmit disease No known test offers complete assurance that they are free of micro organisms Some of the most virulent Hepatitis B and C and HIV I V viruses atypical mycobacteria and certain systemic fungi further emphasize the need for aerosol protection Handle other infectious samples according to good laboratory proce dures and methods to prevent spread of disease Because spills may generate aerosols observe proper safety precautions for aerosol containment Do not run toxic pathogenic or radioactive materials in these rotors without taking appropriate safety precautions Biosafe containment should be used when Risk Group II materials as identified in the World Health Organization Laboratory Biosafety Manual are handled materials of a higher group require more than one level of protection FILLING AND PLUGGING Opt
134. symmetrically on the rotor see Figure 1 6 Opposing tubes must be filled to the same level with liquid of the same density Refer to ROTOR BALANCE in Section 1 III NOTE Place filled tubes in at least two opposing buckets Do not put spacers in buckets that do not contain tubes Ifthe rotor has hook on buckets make certain that both hooks are on the crossbar and that buckets are placed in their proper labeled positions Ifthe rotor has hinge pins lightly lubricate the pin threads with Spinkote Attach each bucket using the hinge pin tool 330069 and 330070 OPERATION For runs at other than room temperature refrigerate or warm the rotor beforehand for fast equilibration INSTALLING THE ROTOR 1 Note the location of the two small indentations on the rotor adapter or the mechanical overspeed devices on older rotors Their position indicates the location of the drive pins Drive Adapter Pins 5 6 Preparative Ultracentrifuge Rotors and Tubes Preparative Ultracentrifuge Rotors and Tubes Using Swinging Bucket Rotors 2 Carefully lift the rotor with both hands do not carry a rotor with hook on buckets by the rotor adapter the buckets may be dislo cated resulting in an unbalanced rotor spilled sample and failed or collapsed tubes and lower it straight down onto the drive hub Make sure that the rotor pins are at a 90 degree angle to the drive hub pins Careful installation will prevent disturbing the sample o
135. t Adapter Removal Tool ca 301875 858122 Figure 3 3 Tools Used to Assemble Tube Caps 1 If possible fill tubes one half to three quarters full before capping Small diameter tubes that use caps without filling holes caps 303624 303658 303113 and 305022 must be completely filled before capping Delrin C pa Nut Washer 2 Loosely assemble the stem the O ring or gasket the crown the crown washer if applicable and the nut The nylon insert should Stem and already be installed in the stem For titanium caps turn the crown Nyon insert slightly to be sure it is properly seated on the stem O ring lt or Gasket 5 Nylon inserts are installed in the stems of cap assemblies purchased as a unit Stems ordered separately do not contain an insert See Section 7 for installation Using Tubes Bottles and Accessories Table 3 4 Required Tools and Torque Values Cap Nut Size Tightening Tool Tube Caps Part Number Torque Value Torque wrench 858121 titanium cap 11 mm 7 16 in 10 to 11 Nem 90 to 100 in Ib Socket 870432 341968 335320 titanium Torque wrench 858121 331151 red 20 mm 3 4 in 11 to 13 6 Nem 100 to 120 in Ib Socket 858122 330901 red 301870 for the first four runs Socket 858123 338905 blue 11 Nem 100 in Ib 338904 black starting with the fifth run 338906 blue 338903 used with 20 mm 3 4 in 11 Nem 100 in Ib Type 21 rotor 301870 Hex driver 841884 30
136. tening tools and torque values 7o avoid strip ping the plugs apply downward pressure to the plug adapter while tightening the plugs The top surface of each rotor plug should be flush with the top surface of the rotor Plugs are not flush on the NVT 65 2 rotor when properly torqued the plugs should protrude not more than 1 mm above the rotor top surface Make sure that all plugs are level with each other BECKMAN Assembly Plug N Rotor Plug Gasket Figure 6 3 Preparing a Vertical Tube or Near Vertical Tube Rotor See Table 6 2 for the correct tightening tools and torque values Preparative Ultracentrifuge Rotors and Tubes OPERATION Preparative Ultracentrifuge Rotors and Tubes Using Vertical Tube and Near Vertical Tube Rotors Table 6 2 Rotor Plugs and Tools Used for Vertical Tube and Near Vertical Tube Rotors Rotors listed in parentheses are no longer manufactured Rotor Torque Plug Plug Wrench Adapter Rotor Part No Part No Part No Torque Value NVT 100 368546 858121 976959 11 Nem 100 in Ib NVT 90 368546 858121 976959 13 6 Nem 120 in Ib VTi 90 368546 858121 976959 13 6 Nem 120 in Ib VTi 80 342881 858121 976959 13 6 Nem 120 in Ib NVT 65 2 368546 858121 976959 13 6 Nem 120 in Ib NVT 65 392084 858121 976959 13 6 Nem 120 in Ib VTi 65 2 368546 858121 976959 13 6 Nem 120 in Ib VTi 65 1 392084 858121 976959 13 6 Nem 120 in Ib VTi 65
137. terial is the more biological material DNA protein cells and so forth will adhere to the walls Appendix F References Documents referenced below can be obtained by calling Beckman Coulter at 1 800 742 2345 in the United States or by contacting your local Beckman Coulter office IN 181 IN 189 IN 192 IN 197 L ML L5 TB 006 L5 TB 010 L5 TB 060 L5 TB 072 L5 TB 081 How to Use Quick Seal Tubes with the Beckman Coulter Cordless Tube Topper Using OptiSeal Tubes Use and Care of Centrifuge Tubes and Bottles Rotor Safety Multi lingual Master Logbook for Ultracentrifuge Rotors Instructions for Using the Tube Cap Vise Instructions for Using the Beckman Tube Slicer Instructions for Using Aluminum Tube Caps in Fixed Angle Ultracentrifuge Rotors Run Speeds for Stainless Steel Tubes Beckman Fraction Recovery Systems For detailed information on a rotor see the applicable individual rotor manual Preparative Ultracentrifuge Rotors and Tubes F 1 References F 2 Documents referenced below are available at www beckmancoulter com or upon request from Beckman Coulter Inc Marketing Communications 4300 N Harbor Blvd Box 3100 Fullerton CA 92834 U S A A 1824 Plasmid Isolation Using NVI Near Vertical Tube Rotor A 1846 Selected Run Conditions for Optimizing the Separation of RNA Using Centrifugation in Either a Preparative Floor or Tabletop Instrument A 1938 Prediction of
138. ternationally contact the U S office to find the dealer closest to you Preparative Ultracentrifuge Rotors and Tubes 7 11 Care and Maintenance e If tube caps are contaminated with toxic or pathogenic solutions c decontaminate or dispose of them as directed by your laboratory e 3 safety officer following appropriate safety guidelines Check the ipe chemical resistances list in Appendix A to be sure the decontami nation method will not damage any part of the rotor STERILIZATION AND DISINFECTION e All cap components except those made of Noryl can be auto claved at 121 C for up to 30 minutes Disassemble caps for autoclaving 121 C Ethanol 7096 6 or hydrogen peroxide 6 may be used on cap components including those made of plastic Bleach sodium hypochlorite may be used but may cause discoloration of anod ized surfaces Use the minimum immersion time for each solution per laboratory standards Guo ur While Beckman Coulter has tested these methods and found that they do not damage components no guarantee of sterility or disinfection is expressed or implied When sterilization or disinfection is a concern consult your laboratory safety officer regarding proper methods to use LUBRICATION Keep the stem threads lightly lubricated with Spinkote lubricant 306812 Clean lubricated threads can be fully tightened without galling or seizing The O ring or gasket must be used dry and without lubric
139. th ethidium bromide The starting density of the CsCl solution is 1 55 g mL In this separation the covalently closed circular plasmid bands at a density of 1 57 g mL while the nicked and linear species band at 1 53 g mL At 20 C where will particles band 1 Gradients in Figure C 2 result from homogeneous CsCl solutions but can be more rapidly generated from step or linear gradients as long as the total amount of CsCl in solution is equal to the amount in the homogeneous solution from the curves in Figure C 1 C4 Preparative Ultracentrifuge Rotors and Tubes Preparative Ultracentrifuge Rotors and Tubes The Use of Cesium Chloride Curves 1 In Figure C 1 find the curve that corresponds to the desired run temperature 20 and tube fill volume full The maximum allowable rotor speed is determined from the point where this curve intersects the homogeneous CsCl density 52 000 rpm In Figure C2 sketch a horizontal line corresponding to each particle s buoyant density Mark the point where each density intersects the curve corre sponding to the maximum speed and selected temperature 4 Particles will band at these points along the tube axis In this example particles will band at about 55 2 and 58 1 mm from the axis of rotation about 2 9 mm of interband center of band to center of band separation at the 25 degree tube angle When the tube is held upright there will be about 3 2 mm of interband separation
140. the rotor cannot be ascertained by Beckman Coulter Use of other manufacturers components or accessories in a Beckman Coulter rotor may void the rotor warranty and should be prohibited by your laboratory safety officer Only the components and accessories listed in the applicable rotor manual should be used TUBES AND BOTTLES Fixed angle rotors can accommodate a variety of tube types listed in the rotor manual Refer to Section 3 for tube filling and sealing or capping requirements Observe the maximum rotor speeds and fill volumes listed in the applicable rotor instruction manual Fill volumes maximum rotor speeds and capping requirements for ultracentrifuge bottles are listed in Section 3 Some rotors must be centrifuged at reduced speeds when bottles are run partially filled Refer to the applicable rotor manual for specific minimum and maximum fill volumes and rotor speeds When running uncapped tubes observe the maximum rotor speeds and fill volumes listed in Table 4 2 ROTOR PREPARATION AND LOADING For runs at other than room temperature refrigerate or warm the rotor beforehand for fast equilibration 4 4 Preparative Ultracentrifuge Rotors and Tubes Using Fixed Angle Rotors Table 4 2 Maximum Run Speeds and Tube Volumes for Uncapped Tubes in Fixed Angle Rotors Maximum Capless Speed Nominal Part Number Maximum rpm Dimensions Volume mm Polycarbonate
141. the vertical tube rotors these rotors have plugs to restrain and support sealed OptiSeal or Quick Seal tubes 6 2 Preparative Ultracentrifuge Rotors and Tubes Using Vertical Tube and Near Vertical Tube Rotors mine rav L max ML Axis of Rotation VTi 65 2 Axis of Rotation VTi 90 Figure 6 1 Vertical Tube Rotors i rav Imax Axis of Rotation NVT 100 Axis of Rotation NVT 65 Figure 6 2 Near Vertical Tube Rotors Preparative Ultracentrifuge Rotors and Tubes 6 3 Using Vertical Tube and Near Vertical Tube Rotors I gt NOTE Although rotor components and accessories made by other manufacturers may fit in the Beckman Coulter rotor you are using their safety in the rotor cannot be ascertained by Beckman Coulter Use of other manufacturers components or accessories in a Beckman Coulter rotor may void the rotor warranty and should be prohibited by your laboratory safety officer Only the components and accessories listed in the applicable rotor manual should be used TUBES AND BOTTLES Only OptiSeal or Quick Seal tubes are used in these rotors Refer to Section 3 for tube filling and sealing or plugging requirements Observe the maximum rotor speeds and fill volumes listed in the applicable rotor instruction manual ROTOR PREPARATION AND LOADING For runs
142. theses are no longer manufactured Radial Distances Number of Relative mm Tubes x Maximum Centrifugal Tube Tube Rotor Speed Field x g Angle k Capacity Type rpm at max degrees Imax lay Imin Factor mL Vertical Tube VTi 90 90 000 645 000 0 71 1 64 5 57 9 6 8x 5 1 VTi 80 80 000 510 000 0 71 1 64 5 57 9 8 8x 5 1 VTi 65 2 65 000 416 000 0 87 9 81 3 74 7 10 16 x 5 1 VTi 65 1 65 000 401 700 0 84 9 76 7 68 5 13 8 x 13 5 VTI 65 65 000 404 000 0 85 4 78 7 72 1 10 8x 5 1 VC 53 53 000 249 000 0 79 0 66 0 53 1 36 8x 39 VTi 50 50 000 242 000 0 86 6 73 7 60 8 36 8x 39 VAC 50 50 000 242 000 0 86 6 73 7 60 8 36 8x39 Near Vertical Tube NVT 100 100 000 750 000 8 67 0 57 6 48 3 8 8 x 5 1 NVT 90 90 000 645 000 8 71 1 61 8 52 4 10 8 x 5 1 NVT 65 2 65 000 416 000 8 5 87 9 78 4 68 8 15 16 x 5 1 NVT 65 65 000 402 000 7 5 84 9 72 2 59 5 21 8 x 13 5 Maximum speeds are based on a solution density of 1 7 g mL in all vertical tube and near vertical tube rotors NEAR VERTICAL TUBE ROTORS Tubes in near vertical tube rotors see Figure 6 2 are held in numbered tube cavities at an angle to the axis of rotation typically 7 to 10 degrees The slight angle of the rotor significantly reduces run times from fixed angle rotors with tube angles of 20 to 35 degrees while allowing components that do not band under separation condi tions to either pellet to the bottom or float to the top of the tube Like
143. tion of sucrose gradients for sedimentation of denatured DNA GRADIENT FORMATION AND FRACTIONATION Consideration should be given to gradient formation and fractionation when choosing a tube for a density gradient run If the bands or zones formed during centrifugation are indistinct they may not be visible through a translucent material such as polyallomer If optimum band visualization is important Ultra Clear polycarbonate or cellulose propionate tubes should be used Whenever collection of bands or zones must be done by slicing or puncturing the tube a thin flexible tube wall is required Ultra Clear or polyallomer tubes should be used in these cases depending on the need for transparency LABWARE TYPES I gt NOTE Tubes made of cellulose nitrate were formerly used for various separations particularly rate zonal separations Beckman Coulter discon tinued the use of cellulose nitrate for tube manufacture in 1980 due to inconsistent physical properties inherent in the material If you currently have cellulose nitrate tubes dispose of them Consult your laboratory safety officer for proper disposal procedures Preparative Ultracentrifuge Rotors and Tubes 2 3 Tubes Bottles and Accessories POLYALLOMER TUBES Open Top Polyallomer Tubes l OptiSeal Tubes C _ Spacer Plug Polyallomer is a copolymer of ethylene and propylene Polyallomer tubes are translucent or transparent in appearance depend
144. trifugation using a combination of short Quick Seal tubes and floating spacers designed to reduce volumes while maximizing separa tion efficiency high density polyethylene used for adapters Preparative Ultracentrifuge Rotors and Tubes isopycnic konical tubes maximum volume mechanical overspeed cartridge meniscus near vertical tube rotor neoprene Noryl OptiSeal tubes overspeed disk pelleting PET polyallomer Preparative Ultracentrifuge Rotors and Tubes Glossary of Terms a method of particle separation or isolation based on particle buoyant density particles are centrifuged until they reach a point in the gradient where the density of the particle is the same as the density of the gradient at that point thin walled polyallomer tubes featuring a conical tip to optimize pelleting separations the conical tip concentrates the pellet in the narrow base of the tube Available in both open top and Quick Seal bell top designs the maximum volume at which a tube should be filled for centrifugation sometimes referred to as maximum fill volume or nominal fill volume an assembly installed in the bases of some older rotors or swinging bucket rotor adapters as part of the mechanical overspeed protection system the curved upper surface of a liquid column that is concave when the container walls are wetted by the liquid and convex when they are not a rotor in which the tubes are held at a slight angle
145. ual to determine the maximum allowable speed and solution density when using partially filled tubes Preparative Ultracentrifuge Rotors and Tubes 1 11 Rotors RATE ZONAL SEPARATIONS Particle separation achieved with rate zonal separation is a function of the particles sedimentation coefficient density size and shape and viscosity of the gradient material Sucrose is especially useful as a gradient material for rate zonal separation because its physical characteristics are well known and it is readily available Samples are layered on top of the gradient Under centrifugal force particles migrate as zones Rate zonal separation is time dependent if the particles are more dense than the most dense portion of the gradient some or all of the particles will pellet unless the run is stopped at the appropriate time A separation is sometimes a combination of rate zonal and isopycnic Depending on particle buoyant densities and sedimentation coeffi cients some particles may be separated by their differential rates of sedimentation while others may reach their isopycnic point in the gradient Clearing factors of swinging bucket rotors at maximum speeds and various particle densities have been calculated for 5 to 2096 wt wt linear sucrose gradients at 5 C These are called k factor and are given in Table 5 1 in Section 5 These constants can be used to estimate the time in hours required to move a zone of particles of known
146. ubes Threads pf Plug Gasket 7 Check for Corrosion M Overspeed Disk Care and Maintenance Regularly check the condition of rotor plugs a component of vertical tube and near vertical tube rotors and rotor plug gaskets Replace worn or damaged gaskets Regularly inspect the overspeed disk If it is scratched damaged or missing replace it FIELD ROTOR INSPECTION PROGRAM LUBRICATION Preparative Ultracentrifuge Rotors and Tubes The Field Rotor Inspection Program FRIP has two purposes to prevent premature rotor failures by detecting conditions such as stress corrosion metal fatigue damage or wear in the anodized coatings and toinstruct laboratory personnel in the proper care of rotors Beckman Coulter has trained a group of experienced service engineers in the techniques of nondestructive evaluation For more information about the program contact your Beckman Coulter representative Proper lubrication is essential to obtain specified torque values where required and to minimize thread wear Many rotors use O rings as seals to maintain atmospheric pressure in the rotor during a run These O rings and the surfaces they bear against must be kept clean and evenly lubricated After removing and cleaning rotor O rings or gaskets lightly but evenly coat them with silicone vacuum grease 335148 and reposition them in the rotor After cleaning metal threads lubri
147. ubes Moisture between the tube and the rotor cavity may lead to tube collapse and increase the force required to extract the tube Slide the tubes into the tube cavities Tubes must be arranged symmetrically in the rotor see Figure 1 6 Opposing tubes must be filled to the same level with liquid of the same density Refer to ROTOR BALANCE in Section 1 Place filled tubes in at least two opposing cavities It is important that each cavity being used is completely filled Use the required spacers and or floating spacers if necessary to complete the loading operation If OptiSeal tubes are being used install a spacer over each plugged tube refer to the applicable rotor manual Leave cavities without tubes completely empty If Quick Seal tubes are being used install spacers and or floating spacers over sealed tubes refer to the applicable rotor manual The particular type of tube support for Quick Seal tubes depends on the length of the tube but the top of the tube must be supported Leave cavities without tubes completely empty A CAUTION To prevent plug damage do not put spacers or plugs in cavities that do not contain tubes Leave unused tube cavities completely empty 6 5 Using Vertical Tube and Near Vertical Tube Rotors 6 6 5 Insert a rotor plug with the white gasket end down over each spacer screw in the plug 6 Tighten each rotor plug as shown in Figure 6 3 Refer to Table 6 2 for the correct tigh
148. ubes and Open the Chemical Resistances Chart Click Here Preparative Ultracentrifuge Rotors and Tubes A 1 Appendix B Use of the wt Integrator The centrifugal force applied to a sample in a spinning rotor is shown by r where r is the radial distance from the axis of rotation and amp is the angular velocity in radians per second o 2x rpm 60 The sedimentation velocity dr dt is proportional to the centrifugal force the velocity of a sedimenting particle increases as it moves outward in the tube Thus a force corrected velocity is used to describe the movement of particles under centrifugal force This is the sedimenta tion coefficient s defined as the sedimentation velocity per unit of centrifugal force x B 1 pe B 2 where r is the initial position of the particle and r is the final position relative to the axis of rotation These distances can be readily determined However an accurate measure of the centrifugal force applied to the particle necessitates that the value of generated during periods of changing speed be calculated that is from the time when the rotor starts spinning t4 until the rotor stops t5 The t integrator automatically computes the total centrifugal effect during acceleration constant speed operation speed changes and deceleration and displays this as a continuously updated value of ty f dt B 3 fi Preparative Ultracentrifuge Rotors and Tubes B 1 Us
149. ugation With the tube plug and spacer and rotor plug if required in place the g forces during centrifugation ensure a tight reliable seal that protects your samples For a detailed discussion on the use of OptiSeal tubes refer to Using OptiSeal Tubes publication IN 189 included with each box of tubes Preparative Ultracentrifuge Rotors and Tubes Quick Seal Polyallomer Tubes g Max Metal Floating Spacer Spacer oM A Dome Top Bell Top POLYCARBONATE TUBES 3 POLYPROPYLENE TUBES Preparative Ultracentrifuge Rotors and Tubes Tubes Bottles and Accessoires Heat sealed Quick Seal tubes are used in swinging bucket vertical tube near vertical tube and in most fixed angle rotors Single use Quick Seal tubes are a convenient form of sealable tube they are especially useful for the containment of radioactive or pathogenic samples There are two Quick Seal tube designs dome top and bell top The bell top simplifies removal of materials that float during centrifugation Dome top tubes hold more volume than their bell top equivalents Detailed information about Quick Seal tubes is contained in publication IN 181 Polycarbonate is tough rigid nonwettable and glass like in appear ance Polycarbonate tubes are used with or without caps in fixed angle rotors and at least half full in swinging bucket rotors Speed reduction may be required in some rotors if the tubes are not completely filled Althou
150. uge Rotors and Tubes DESCRIPTION Preparative Ultracentrifuge Rotors and Tubes 4 Using Fixed Angle Rotors This section contains instructions for using fixed angle rotors in preparative ultracentrifuges In addition to these instructions observe procedures and precautions provided in the applicable rotor and ultracentrifuge manuals Refer to Section 2 for labware selection information and Section 3 for recommended filling and sealing or capping requirements and for sample recovery procedures Refer to Section 7 for information on the care of rotors and accessories Fixed angle rotors see Figure 4 1 are general purpose rotors that are especially useful for pelleting and isopycnic separations Refer to Table 4 1 for general rotor specifications Tubes in fixed angle rotors are held at an angle usually 20 to 35 degrees to the axis of rotation in numbered tube cavities The tube angle shortens the particle pathlength compared to swinging bucket rotors resulting in reduced run times Most fixed angle rotors have a lid secured by a handle Most handles have holes so that a screwdriver or metal rod can be used to loosen the lid after centrifugation The lids of some high performance rotors have either two or four small holes to provide a temporary vent which prevents rotor damage by allowing liquid to escape in the event of tube leakage O rings made of Buna N rubber are located in the rotor lid The O rings help to maintain
151. uge Rotors and Tubes Table D 4 Density Conversion for Cesium and Rubidium Salts at 20 C Gradient Materials w w CsCl CsBr Csl Cs S0 CsNO3 RbCI RbBr Rbi RbSO4 RbNOs 1 1 00593 1 00612 1 00608 1 0061 1 00566 1 00561 1 00593 1 00591 1 0066 1 0053 2 1 01374 1 01412 1 01402 1 0144 1 01319 1 01307 1 01372 1 01370 1 0150 1 0125 4 1 02969 1 03048 1 03029 1 0316 1 02859 1 02825 1 02965 1 02963 1 0322 1 0272 6 1 04609 1 04734 1 04707 1 0494 1 04443 1 04379 1 04604 1 04604 1 0499 1 0422 8 1 06297 1 06472 1 06438 1 0676 1 06072 1 05917 1 06291 1 06296 1 0680 1 0575 10 1 08036 1 08265 1 08225 1 0870 1 07745 1 07604 1 08028 1 08041 1 0864 1 0731 12 1 09828 1 10116 1 10071 1 1071 1 09463 1 09281 1 09817 1 09842 1 1052 1 0892 14 1 11676 1 12029 1 11979 1 1275 1 11227 1 11004 1 11661 1 11701 1 1246 1 1057 16 1 13582 1 14007 1 13953 1 1484 1 12775 1 13563 1 13621 1 1446 1 1227 18 1 15549 1 16053 1 15996 1 1696 1 14596 1 15526 1 15605 1 1652 1 1401 20 1 17580 1 18107 1 18112 1 1913 1 16469 1 17554 1 17657 1 1864 1 1580 22 1 19679 1 20362 1 20305 1 2137 1 18396 1 19650 1 19781 1 2083 1 1763 24 1 21849 1 22634 1 22580 1 2375 1 20379 1 24817 1 21980 1 2309 1 1952 26 1 24093 1 24990 1 24942 1 264
152. ures are too harsh Wash tubes bottles adapters and other accessories by hand using a mild detergent such as Solution 555 339555 diluted 10 to 1 with water and a soft brush e Polycarbonate bottles and tubes are vulnerable to attack by alkaline solutions and detergents so use a detergent with pH less than 9 such as Solution 555 Do not use a brush with exposed metal scratches in polycarbonate will cause early failure Preparative Ultracentrifuge Rotors and Tubes Care and Maintenance Alcohol and acetone react unsatisfactorily with many tube and accessory materials If a solvent must be used to rinse dry or decontaminate these materials consult Appendix A to select an appropriate solvent Do not dry tubes bottles or accessories in an oven Labware should be air dried e OptiSeal Quick Seal Ultra Clear and thinwall polyallomer tubes are intended for one time use and should be discarded after use DECONTAMINATION Labware contaminated with radioactive or pathogenic solutions should be decontaminated or disposed of following appropriate safety guidelines and or regulations Consult Appendix A to select an agent that will not damage the tube or bottle material STERILIZATION AND DISINFECTION 121 C Eo gt Preparative Ultracentrifuge Rotors and Tubes Refer to Table 7 1 for sterilization methods recommended for each container type Most tubes and accessories except those made of Ultra Clear p
153. used Refer to Appendix A Verify that tubes bottles and accessories being used are listed in the appropriate rotor manual ROTOR PREPARATION AND LOADING 4 6 1 Be sure that metal threads in the rotor are clean and lightly but evenly lubricated with Spinkote lubricant 306812 Also ensure that O rings are lightly but evenly coated with silicone vacuum grease 335148 Dry the exterior of the tubes Moisture between the tube and the rotor cavity may lead to tube collapse and increase the force required to extract the tube Slide the filled and capped or sealed tubes into the tube cavities Tubes must be arranged symmetrically in the rotor see Figure 1 6 Opposing tubes must be filled to the same level with liquid of the same density Refer to ROTOR BALANCE in Section 1 gt NOTE Place filled tubes in at least two opposing cavities Make sure that cavities in use also have the proper spacers inserted before installing the rotor lid Do not put spacers in cavities that do not contain tubes Preparative Ultracentrifuge Rotors and Tubes 4 Dome Top Tube Spacer Tube ER Pug Tube Floating Spacer Bell Top Tube C OPERATION Preparative Ultracentrifuge Rotors and Tubes Using Fixed Angle Rotors 3 Use the required spacers and or floating spacers if necessary to complete the loading operation If Opt
154. usually 7 to 10 degrees black synthetic elastomer used for O rings in some tube caps and bottle cap assemblies should be used at temperatures between 54 and 121 C 65 and 250 F modified thermoplastic polyphenylene oxide PPO used for floating spacers part of the g Max system and some polycarbonate bottle caps Norylis a registered trademark of GE Plastics capless tubes with sealing plugs inserted in the tube stems during centrifu gation the combination of g force and hydrostatic pressure seals the tube an adhesive disk with alternating reflecting and nonreflecting sectors attached to the bottom of rotors as part of the photoelectric overspeed protec tion system the number of sectors on the disk is a function of the rotor s maximum allowable speed a centrifugal separation process in which particles in a sample sediment to the bottom of the tube differential separation differential pelleting sepa rates particles of different sizes by successive centrifugation steps of progressively higher g force and or longer run duration polyethylene terephthalate used in some adapters random block copolymer of ethylene and propylene used for certain tubes Tenite Polyallomer is a registered trademark of Eastman Chemical Co Glossary of Terms Quick Seal tubes Radel rate zonal RCF Tmax Tmin sedimentation sedimentation coefficient s silicone rubber Solution 555 bell top or dome top thinwall tub
155. vely lighter concentrations This method is acceptable for wettable tubes however loading a nonwettable tube such as Ultra Clear polyallomer and polycarbonate by allowing solutions to run down the side of the tube can cause mixing 10 15 Added Gradients in nonwettable tubes can be prepared using a gradient Last oat 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 A complete list of tubes bottles and adapters is provided in the latest edition of the Beckman Coulter Ultracentrifuge Rotors Tubes amp 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 Preparative Ultracentrifuge Rotors and Tubes 3 1 Using Tubes Bottles and Accessories 1 mL Syringe Bee 20 to 22 Gauge Needle g P 2 to 3mm ze t Gradient 2 to 3 mm Buffer Sample d Gradient 210 3 Sucrose Added You can also prepare preformed step gradients by hand using a pipette
156. y to ensure that the parts are free from pathogens chemical hazards and or radio activity Sterilization and decontamination MUST be done before returning the parts Smaller items such as tubes bottles and so on should be enclosed in a sealed plastic bag All parts must be accompanied by a note plainly visible on the outside of the box or bag stating that they are safe to handle and that they are not contaminated with pathogens chemical hazards or radioactivity Failure to attach this notification will result in return or disposal of the items without review of the reported problem Use the address label printed on the RGA form when mailing the rotor and or accessories to Beckman Coulter Inc Spinco Business Center 1050 Page Mill Road Palo Alto CA 94304 Attention Returned Goods Customers located outside the United States should contact their local Beckman Coulter office 7 14 Preparative Ultracentrifuge Rotors and Tubes DIAGNOSTIC HINTS SYMPTOM Rotors Severe vibration Stripped rotor plugs on vertical tube or near vertical tube rotors Rotor lid is difficult to remove after centrifugation Paint coming off where bucket contacts rotor pocket on swinging bucket rotor Preparative Ultracentrifuge Rotors and Tubes Care and Maintenance Some of the more common operating problems experienced in centrifugation are listed below with suggestions for their solutions Contact Beckman Coulter Field Service if a pr

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