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Operating Manual

1. 1 90 1 85 1 80 NS ny N 1 75 1 70 a 1 69 ZTN The dashed line is a representation of the 1 60 En equation a 2 g 155 2 RPM 90 000 _1 2 g mL 5 p e 1 50 B 5 8 145 E where p density of s i tube contents g a 1 40 D and is shown here to illustrate the inability 1 35 E of that equation to D guard against CsCl 1 30 2 precipitation 1 25 TYPE 90 Ti ROTOR 1 20 D Zhen 26 1 15 1 10 1 05 1 00 0 10 20 30 40 50 60 70 80 90 Rotor Speed K rpm Using combinations of rotor speeds and homogeneous CsCl solution densities that intersect on or below these curves ensures that CsCl will not precipitate during centrifugation C 2 LR IM 24AC The Use of Cesium Chloride Curves C Cesium Chloride Curves Figure C 2 CsCl Gradients at Equilibrium 1 90 1 85 TYPE 90 Ti ROTOR 1 80 20 C 4 C 1 75 a 1 70 1
2. 1 Make sure that the rotor and lid are clean and show no signs of corrosion or cracking 2 Make sure the rotor is equipped with the correct overspeed disk refer to CHAPTER 1 If the disk is missing or damaged replace it as described in CHAPTER 7 ofr sg 60 un 30 Sector E W 331155 gt aar 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 a Rotor Preparation and Loading 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 5 4 LR IM 24AC LR IM 24AC Using Swinging Bucket Rotors Rotor Preparation and Loading 3 Remove the bucket gaskets or O rings and coat them lightly but evenly with silicone vacuum grease 335148 a Install gaskets or O rings in the buckets Eca 1 Gasket Bucket 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 e Moisture between the tube and the bucket may lead to tube collapse and increase the force required to extract the tube a Slide the filled and sealed tubes into the buckets Loaded buckets can be supported in the bucket holder rack available for
3. Table 6 2 Rotor Plugs and Tools Used for Vertical Tube and Near Vertical Tube Rotors 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 lb VTi 90 368546 858121 976959 13 6 Nem 120 in lb VTi 80 342881 858121 976959 13 6 Nem 120 in lb NVT 65 2 368546 858121 976959 13 6 Nem 120 in lb NVT 65 392084 858121 976959 13 6 Nem 120 in lb VTi 65 2 368546 858121 976959 13 6 Nem 120 in lb VTi 65 1 392084 858121 976959 13 6 Nem 120 in Ib VTi 65 355874 858121 976959 10 to 11 Nem 90 to 100 in Ib VC 53 355587 889096 355588 17 5 Nem 150 in lb VTi 50 355587 889096 355588 17 5 Nem 150 in Ib VAC 50 355587 889096 355588 17 5 Nem 150 in Ib a Rotors listed in parentheses are no longer manufactured b Part number 858121 is a 1 4 in drive torque wrench part number 889096 is a 3 8 in drive torque wrench c The VTi 50 and VC 53 rotors and rotor plugs must be cooled or warmed to operating temperature before torquing or leakage may result 6 8 LR IM 24AC Using Vertical Tube and Near Vertical Tube Rotors 6 Operation Removal and Sample Recovery 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
4. Sterilization and Disinfection 121 C CI u All cap components except those made of Noryl can be autoclaved at 121 C for up to 30 minutes Disassemble caps for autoclaving Ethanol 70 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 anodized surfaces Use the minimum immersion time for each solution per laboratory standards 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 7 12 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 lubrication 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 In U S contact Biodex Medical Systems Shirley New York internationally contact the U S office to find the dealer closest to you t 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
5. cap Polypropylene 3 3 Using Tubes Bottles and Accessories Filling and Plugging OptiSeal Tubes Table 3 1 Filling and Capping Requirements for Tubes and Bottles Continued Filling Level Requirements Vertical and Near Swinging Bucket Vertical Tube Tubes or Bottles Rotors Fixed Angle Rotors Rotors 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 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 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 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 OptiSeal Tubes OptiSeal tubes are not sealed prior to centrifugation a Noryl
6. Teflon is a registered trademark of E I Du Pont de Nemours amp Co 3 10 LR IM 24AC Using Tubes Bottles and Accessories Filling and Sealing Quick Seal Tubes Method A With the Seal Guide Seal Guide vz y Heat Sink LR IM 24AC a Place a seal guide with the flat side down over the seal former Turn the Tube Topper pushbutton to USE position Press the pushbutton and wait 3 to 5 seconds for the tip to heat 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 NOTE Always apply the tip of the Tube Topper vertically to the seal former Apply gentle pressure when sealing the tube 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 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 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 11 3 Using Tubes Bottles and Accessories Filling and
7. O Ring 2 Rinse thoroughly with water 3 Air dry the body or buckets upside down a Do not use acetone to dry rotors 4 Wipe clean the O rings or gaskets regularly lubricate after cleaning a Replace them about twice a year or as required 7 2 LR IM 24AC Care and Maintenance Rotor Care 5 Frequently clean all surfaces that contact O rings a 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 b Lubricate the threads as directed under Lubrication below Decontamination 121 C Q D LR IM 24AC Rh age Rotors contaminated with radioactive or pathogenic materials must be decontaminated following appropriate laboratory safety guidelines and or other regulations NOTE Strong bases and or high pH solutions can damage aluminum rotors and components If a rotor and or accessories becomes contaminated with radioactive 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 NOTE IsoClean can cause fading of colored anodized surfaces Use it only when necessary and do not soak rot
8. Opposing tubes must be filled to the same level with liquid of the same density Refer to Rotor Balance in CHAPTER 1 a 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 b Ifthe rotor has hinge pins lightly lubricate the pin threads with Spinkote 1 Attach each bucket using the hinge pin tool 330069 and 330070 5 6 LR IM 24AC Using Swinging Bucket Rotors 5 Operation NOTE Place filled tubes in at least two opposing buckets Do not put spacers in buckets that do not contain tubes Operation For runs at other than room temperature refrigerate or warm the rotor beforehand for fast equilibration 1 Note the location of the two small indentations on the rotor adapter or the mechanical overspeed devices on older rotors e Their position indicates the location of the drive pins rive Adapter Pins LR IM 24AC 5 7 Using Swinging Bucket Rotors Operation 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 dislocated resulting in an unbalanced rotor spilled sample and failed or collapsed tubes and lower it straight down onto the drive hub a Make sure that the rotor pins are at a 90 degree angle to the drive hub pins e Careful installation will prevent disturbing the sample or tripping the imbalance detector If hook on bucket
9. 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 28 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 a To the maximum speed of the ultracentrifuge as applicable Special Action on Older Type 35 and Type 42 1 Rotors xvi We have found that there is a
10. ge Spacer ra lube Plug _7 Tube c If Quick Seal tubes are being used install spacers and or floating spacers over sealed tubes refer to the applicable rotor manual e 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 4 6 LR IM 24AC Using Fixed Angle Rotors Operation d Leave cavities without tubes completely empty Metal Spacers aL gt 2 Floating l Spacer L 4 Place the lid on the rotor and tighten it as firmly as possible with the handle Dome Top Tube Bell Top Tube a Screw the handle down clockwise to fully compress the O rings 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 CHAPTER 3 Check the tube cavity for foreign matter Operation For runs at other than room temperature refrigerate or warm the rotor beforehand for fast equilibration Installing the Rotor 1 Carefully lower the rotor straight down onto the drive hub LR IM 24AC 4 7 4 Using Fixed Angle Rotors Operation a Ifthe rotor has drive pins install it so that the pins are at a 90 degree angle to the pins in the drive hub e Careful installation will prevent disturbing the sample or tripping the imbalance detector b Refer to the ce
11. Itshould not be corrosive to the rotor Itshould 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 LR IM 24AC D 1 Gradient Materials Description Table D 1 Commonly Used Gradient Materials with Their Solvents Maximum Density Materials Solvent at 20 C Sucrose 66 H20 1 32 Sucrose 65 D0 1 37 Silica sols H20 1 30 Diodon H20 1 37 Glycerol H20 1 26 Cesium chloride H20 1 91 D0 1 98 Cesium formate H20 2 10 Cesium acetate H20 2 00 Rubidium chloride H20 1 49 Rubidium formate H20 1 85 Rubidium bromide H20 1 63 Potassium acetate H20 1 41 Potassium formate H20 1 57 D0 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 H20 1 17 Metrizamide H20 1 46 D 2 LR IM 24AC Gradient Materials Description Table D 2 Density Refractive Index and Concentration Data Cesium Chloride at 25 C Molecular Weight 168 372 Density Refractive by mg mL of Density Refractive 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
12. REQUIRED LR IM 24AC www beckmancoulter com Osoon COULTER 2014 Beckman Coulter Inc All Rights Reserved
13. 2 Ti 50 2 Ti 50 Ti 50 45 Ti 42 1 40 35 21 341969 335320 335319 338864 302312 858046 341969 thinwallPP 90 Ti 80 Ti 75 Ti 70 1 Ti 870380 341969 UC 90 Ti 80 Ti 75 Ti 70 1 Ti 3 16 LR IM 24AC Using Tubes Bottles and Accessories 3 Capping Tubes Table 3 3 Tube Cap Assemblies for Open Top Tubes in Fixed Angle Rotors Continued O ring Tube Cap Hex Set or Assembly Nut Crown screw Insert Gasket Stem Tube Type Rotor Type 25 mm 1 in 302359 301870 302169 338864 302312 301473 302168 thinwall PP 30 UC 338904 330791 3389124 338864 302312 878188 338908 thickwallPP 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 331153h 338864 302312 334280 331152 thinwall PP 70 Ti 60 Ti 55 2 Ti UC 50 2 Ti 42 1 338906 330791 3399 5h 338864 302312 878188 338908 thickwallPP 70 Ti 60 Ti 55 2 Ti PC 50 2 Ti 42 1 3379279 330791 338863 338864 302312 338865 thinwall PP 70 Ti 38 mm 1 1 2 in 32681 301870 326890 808482 302312 346242 326889 thinwall PP 21 UC 338903 330791 3389144 338864 302312 341767 338909 thickwallPP 21 PC 326905 301870 326890 338864 302312 801761 326899 SS 45 Ti 35 21 330901 330791 339793h 338864 302312 346242 330900 thinwall PP 45 Ti 35 UC 338905 330791 3389139
14. 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 32407 1 35191 1 35598 1 3281 1 2764 40 1 42245 1 44275 1 44354 1 38599 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 LR IM 24AC D 5 D Gradient Materials Description D 6 LR IM 24AC APPENDIX E References List of References Documents referenced below can be obtained at www beckmancoulter com or by calling Beckman Coulter at 1 800 742 2345 in the United States or by contacting your local Beckman Coulter office IN 181 How to Use Quick Seal Tubes with the Beckman Coulter Cordless Tube Topper IN 189 Using OptiSeal Tubes IN 192 Use and Care of Centrifuge Tubes and Bottles IN 197 Rotor Safety Multi lingual L ML Master Logbook for Ultracentrifuge Rotors L5 TB 006 Instructions for Using the Tube Cap Vise L5 TB 010 Instructions for Using the Beckman Tube Slicer L5 TB 060 Instructions for Using Aluminum Tube Caps in Fixed Angle Ultracentrifuge Rotors L5 TB 072 Run Speeds for Stainless Steel Tubes L5 TB 081 Beckman Fraction Recovery Systems Documents referenced below are available upon request from Beckman Coulter Inc Marketing
15. 338864 302312 341767 338909 thickwall PP 45 Ti 35 PC a Tube caps are not available b Tube caps are aluminum unless otherwise noted c Abbreviations PP polypropylene PC polycarbonate SS stainless steel UC UltraClear d Aluminum and stainless steel e ube cap is optional Use a tube cap when centrifuging a thickwall tube at its maximum fill capacity f Titanium g Aluminum and titanium h Washer part number 330899 is also required 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 polypropylene 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 LR IM 24AC Using Tubes Bottles and Accessories Capping Tubes 3 18 Blue anodized Aluminum caps with blue anodized crowns are used with thickwall polypropylene and polycarbonate tubes for centrifugation at their maximum fill volumes in high performance rotors The cap nut should be tightened with a torque wrench while the tube is held in the tube cap vise Clear and black anodized 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
16. 35 34 21 000 15 000 21 a Maximum speeds are those for capless tubes tested at 25 C for 24 hours b Rotors are not listed for tubes used with adapters c Cellulose propionate Rotor Preparation and Loading LR IM 24AC lubricant 306812 a Also ensure that O rings are lightly but evenly coated with silicone vacuum grease 335148 1 Be sure that metal threads in the rotor are clean and lightly but evenly lubricated with Spinkote 4 5 4 Using Fixed Angle Rotors Rotor Preparation and Loading 2 Dry the exterior of the tubes e Moisture between the tube and the rotor cavity may lead to tube collapse and increase the force required to extract the tube a Slide the filled and capped or sealed tubes into the tube cavities Tubes must be arranged symmetrically in the rotor see Figure 1 5 Opposing tubes must be filled to the same level with liquid of the same density Refer to Rotor Balance in CHAPTER 1 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 3 Use the required spacers and or floating spacers if necessary to complete the loading operation a If OptiSeal tubes are being used install a spacer over each plugged tube refer to the applicable rotor manual b Leave cavities without tubes completely empty
17. 7 16 LR IM 24AC Care and Maintenance Diagnostic Hints Table 7 2 Troubleshooting Chart Continued Symptom Possible Cause and Suggested Action Bottle leakage uncapped bottles Bottle too full the meniscus must be kept lower to prevent leakage Refer to the rotor manual for fill volumes and speed reductions Bottle damage 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 temperature limits evaluate them under centrifugation conditions If sample is frozen in bottles make sure that bottles are thawed to at least 2 C 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 LR IM 24AC 7 17 Care and Maintenance Diagnostic Hints 7 18 LR IM 24AC APPENDIX A Chemical Resistances for Beckman Coulter Centrifugation Products
18. 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 Ifa Beckman Coulter centrifuge is damaged due to a failure of a rotor covered by this warranty Beckman Coulter will supply free of charge i 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 Warranty 1 Ultracentrifuge Rotor Warranty Warranty 2 the centrifuge is currently covered by a Beckman Coulter warranty or Full Service Agreement all labor necessary for repair of the centrifuge 3 Ifa Beckman Coulter rotor covered by this warranty is damaged 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 Ifa Beckman Coulter rotor covered by this warranty is damaged 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 malfu
19. 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 1984 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 essential that you use this program to determine which rotors may be safely run in which instruments Rotors in parentheses are no longer manufactured 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 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 5
20. Program FRIP This program involves a visit to LR IM 24AC LR IM 24AC Safety Notice Mechanical Safety your laboratory by a specially trained Beckman Coulter 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 office to request this service It is your responsibility to decontaminate the rotors and accessories before requesting service by Beckman Coulter Field Service vi Safety Notice Mechanical Safety LR IM 24AC Contents _ Safety Notice iii Scope xiii Classification Program xv CHAPTER 1 Rotors 1 1 Introduction 1 1 General Description 1 1 Rotor Designations 1 1 Materials 1 2 Drive Pins 1 3 Rotor Selection 1 3 Pelleting Differential Separation 1 8 Isopycnic Separations 1 11 Rate Zonal Separations 1 13 General Operating Information 1 14 Rotor Balance 1 15 Overspeed Protection 1 15 Allowable Run Speeds 1 17 CHAPTER 2 Tubes Bottles and Accessories 2 1 Introduction 2 1 Labware Selection Criteria 2 1 Labware Material Compatibility with Solvents and Sample 2 2 Gradient Formation and Fractionation 2 3 Labware Types 2 3 Polypropylene Tubes 2 3 Open Top Polypropylene Tubes 2 3 OptiSeal Tubes 2 4 Quick Seal Polypropylene Tubes 2 4 Polycarbonate Tubes 2 4 Polypropylene Tubes 2 5 Polyethy
21. Rotor Care 4 Regularly check the condition of rotor plugs a component of vertical tube and near vertical tube rotors and rotor plug gaskets a Replace worn or damaged gaskets Threads Plug Gasket 7 Check for Corrosion N Overspeed Disk 5 Regularly inspect the overspeed disk a Ifit is scratched damaged or missing replace it Field Rotor Inspection Plan 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 to instruct 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 Lubrication Proper lubrication is essential to obtain specified torque values where required and to minimize thread wear e 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 lubricate them with Spinkote lubricant 306812 Failure to keep threads properly
22. Separations Sample Loading 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 Ultracentrifuge Datasheet Optima L 100 XP Ultracentrifuge Datasheet OptiSeal Ultracentrifuge Tubes Rapid Isolation of Both RNA amp DNA from Cultured Cell Purity Antigenicity and Immunogenicity LR IM 24AC Angular velocity Rate of rotation measured in radians per second _ 2mrpm o oO or 0 10472 rom Anodized coating A thin hard layer of aluminum oxide formed electrochemically on aluminum rotor and or accessory surfaces as a protective coating for corrosion resistance Autoclaving Sterilization by heat dry or steam Buoyant density The density of a particle ina specified liquid medium Buna N 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 Centrifugal effect Accumulated value of t3 o dt t1 where t is time and is angular velocity Centrifugal force In a centrifugal field the force which causes a particle to move away from the center of rotation Clearing factor k Calculated for all Beckman Coulter high speed rotors as a measure of the rotor s relative p
23. Spacers are shown in the correct orientation for placement onto tubes b Disposible plastic plugs included c Ultem is a registered trademark of GE Plastics 3 6 LR IM 24AC Figure 3 1 Filling OptiSeal Tubes Seating the Tube Plugs NOTE Meniscus may not be symmetrical Using Tubes Bottles and Accessories 3 Filling and Plugging OptiSeal Tubes Stem xN Base leal NI Meniscus Regular Top Stem Base lt lt Ls y ON Meniscus Between Lines Shown Bell Top Eight tubes can be prepared for use at once in the specially designed racks listed in Table 3 2 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 Low 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 Stems are large enough to accept standard pipettes LR IM 24AC 3 7 Using Tubes Bottles and Accessories Filling and Sealing Quick Seal Tubes 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 se
24. 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 polypropylene 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 alighter 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 imbalance 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 LR IM 24AC Inspecting and Lubricating Tube Caps Inspect Here Tube Cap Crown Inspect Here Setscrew Insert Assembling Tube Caps Using Tubes Bottles and Accessories Capping Tubes 1 Inspect cap components before each use Replace any damaged components Inspect the cap crown for stress cracking and che
25. Ti Rotor B 3 Precipitation Curves for the Type 90 Ti Rotor C 2 CsCl Gradients at Equilibrium C 3 xi Tables xii Tables 1 1 Balk 2 2 Za 3 1 cre 3 3 3 4 3 5 4 1 4 2 5 1 6 1 6 2 7 1 Fa D 1 D 2 D 3 D 4 Beckman Coulter Preparative Rotors by Use 1 6 Characteristics and Chemical Resistances of Tube and Bottle Materials 2 2 Quick Seal Tube Spacers 2 8 Dimensions of Delrin Adapters 2 10 Filling and Capping Requirements for Tubes and Bottles 3 3 OptiSeal Tubes and Accessories 3 5 Tube Cap Assemblies for Open Top Tubes in Fixed Angle Rotors 3 16 Required Tools and Torque Values 3 20 Available Bottles Assembly and Operation 3 24 General Specifications for Beckman Coulter Preparative Fixed Angle Rotors 4 3 Maximum Run Speeds and Tube Volumes for Uncapped Tubes in Fixed Angle Rotors 4 4 General Specifications for Beckman Coulter Preparative Swing ing Bucket Rotors 5 3 General Specifications for Beckman Coulter Preparative Verti cal Tube and Near Vertical Tube Rotors 6 2 Rotor Plugs and Tools Used for Vertical Tube and Near Vertical Tube Rotors 6 8 Tube and Bottle Sterilization and Disinfection 7 9 Troubleshooting Chart 7 15 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 Molec
26. Tube collapse e 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 e 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 e Reagent used that attacks the tube material Refer to APPENDIX A for chemical compatibilities of tube material and chemicals e Tubes run above their rated speed Refer to the applicable rotor manual for maximum speeds Tube Caps Unsure of cap components For a complete list of cap components see the Beckman Coulter Ultracentrifuge Rotors Tubes amp Accessories catalog publication BR 8101 available at www beckmancoulter com Setscrew is difficult to remove The hex socket or threads of the screw may be stripped If the screw cannot be removed replace the cap stem Setscrew will not seal the tube cap Replace the screw and nylon insert if either seems damaged or loose Bottles Bottle leakage bottles with cap assemblies e Moisture or lubrication on cap or sealing surface Ensure that 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 Sealing surface of the bottle is not smooth Replace bottle
27. Using Tubes Bottles and Accessories Filling Open Top Tubes Filling Open Top Tubes Open Top Polypropylene Tubes Open top polypropylene 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 Ww proper tube wall support e 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 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 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 e For swinging bucket rotors fill to within 2 or 3 mm of the top of the tube LR IM 24AC
28. _ Introduction This chapter describes various labware used in Beckman Coulter preparative rotors General instructions for using containers follow in CHAPTER 3 Care and maintenance instructions are in CHAPTER 7 General rotor use instructions are in CHAPTER 4 CHAPTER 5 and CHAPTER 6 The individual 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 e Chemical resistance the nature of the sample and any solvent or gradient media e 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 LR IM 24AC 2 1 Tubes Bottles and Accessories Labware Selection Criteria Table 2 1 Characterist
29. are unsuccessful 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 e Ifanuncapped 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 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 Tube Cap Care 7 10 It is 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 components 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 LR IM 24AC Care and Maintenance Tube Cap Care Cleaning 1 Disassemble tube caps and wash them in a mild detergent solution such as Beckman Solution 555 339555 diluted 10 to 1 with water a If necessary scrub the inside of caps using a cotton tipped swab or a brush that will not scratch the surface NOTE Do not soak aluminum cap parts in a s
30. arranged symmetrically in the rotor see Figure 1 5 Opposing tubes must be filled to the same level with liquid of the same density Refer to Rotor Balance in CHAPTER 1 b Place filled tubes in at least two opposing cavities 4 itis important that each cavity being used is completely filled Use the required spacers and or floating spacers if necessary to complete the loading operation a If OptiSeal tubes are being used install a spacer over each plugged tube refer to the applicable rotor manual LR IM 24AC 6 5 Using Vertical Tube and Near Vertical Tube Rotors Rotor Preparation and Loading 1 Leave cavities without tubes completely empty Ema Spacer Tube mE Pug _ Tube LY b If Quick Seal tubes are being used install spacers and or floating spacers over sealed tubes refer to the applicable rotor manual Spacers N Floating ey Spacer Bell Top Tube Dome Top Tube la w 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 1 Leave cavities without tubes completely empty To prevent plug damage do not put spacers or plugs in cavities that do not contain tubes Leave unused tube cavities completely empty 5 Insert a rotor plug with the white gasket end down over each spacer screw in the plug 6 6 LR IM 24AC Using Vertical Tube and Near V
31. ee Appendix A is replicated in the separate pdf document Chemical Resistances IN 175 LR IM 24AC A 1 Chemical Resistances for Beckman Coulter Centrifugation Products A 2 LR IM 24AC APPENDIX B Use of the wt Integrator _ wt Integrator The centrifugal force applied to a sample in a spinning rotor is shown by wr where r is the radial distance from the axis of rotation and is the angular velocity in radians per second 2m 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 sedimentation coefficient s defined as the sedimentation velocity per unit of centrifugal force dr 1 x B 1 S dt or Pa The integrated form of the equation is r in 5 B 2 at where ry 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 w generated during periods of changing speed be calculated that is from the time when the rotor starts spinning t until the rotor stops t The wt integrator automatically computes the total centrifugal effect during acceleration constant speed operati
32. 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 LR IM 24AC CHAPTER 1 Rotors _ Introduction This chapter 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 CHAPTER 4 CHAPTER 5 and CHAPTER 6 Care and maintenance information for all of these rotors is contained in CHAPTER 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 LR IM 24AC Beckman Coulter preparative rotors are named according to the type of rotor the material composition and the rotor s maximum allowable 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 possi
33. in the applicable rotor manual should be used 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 conditions to either pellet to the bottom or float to the top of the tube Like the vertical tube rotors these rotors have plugs to restrain and support sealed OptiSeal or Quick Seal tubes 6 2 LR IM 24AC Figure 6 1 Vertical Tube Rotors min _ lav H max _ Axis of Rotation ett A __lav a Tmax Axis of Rotation VTi 65 2 VTi 90 Figure 6 2 Near Vertical Tube Rotors Axis of Rotation Axis of Rotation l LR IM 24AC NVT 100 NVT 65 Using Vertical Tube and Near Vertical Tube Rotors Description 6 3 6 Using Vertical Tube and Near Vertical Tube Rotors Tubes and Bottles Tubes and Bottles Only OptiSeal or Quick Seal tubes are used in these rotors Refer to CHAPTER 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 a
34. in two columns read the figure where the straightedge intersects the third column 1 12 LR IM 24AC Rotors 1 Rotor Selection 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 pp Pe 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 manual to determine the maximum allowable speed and solution density when using partially filled tubes 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 a
35. 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 centrifuged at any temperature N a o O O 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 2 6 LR IM 24AC Tubes Bottles and Accessories Spacers and Floating Spacers Spacers and Floating Spacers Floating Spacer Adapters A i Spacer Adapters OptiSeal tubes must be used with the appropriate spacer to seal properly OptiSeal spacers are listed in Table 3 2 Quick Seal tubes use a spacer Table 2 2 one or more floating 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 th
36. 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 Do not use flammable substances in or near operating centrifuges 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 preparation of sucrose gradients for sedimentation of denatured DNA 2 2 LR IM 24AC Tubes Bottles and Accessories 2 Labware Types 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 polypropylene 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 tu
37. oxide PPO used for floating spacers part of the g Max system and some polycarbonate bottle caps Noryl is a registered trademark of GE Plastics OptiSeal tubes Capless tubes with sealing plugs inserted in the tube stems during centrifugation the combination of g force and hydrostatic pressure seals the tube Overspeed disk An adhesive disk with alternating reflecting and nonreflecting sectors attached to the bottom of rotors as part of the photoelectric overspeed protection system the number of sectors on the disk is a function of the rotor s maximum allowable speed Pelleting A centrifugal separation process in which particles in a sample sediment to the bottom of the tube differential separation differential pelleting separates particles of different sizes by successive centrifugation steps of progressively higher g force and or longer run duration PET polyethylene terephthalate used in some adapters Polypropylene Random block copolymer of ethylene and propylene used for certain tubes Tenite Polypropylene is a registered trademark of Eastman Chemical Co Quick Seal tubes bell top or dome top thinwall tubes that are heat sealed and require no caps Radel Polyphenylsulfone PPS used in plugs cap closures cannisters and other accessories Rate zonal A method of particle separation based on differential rate of sedimentation using a preformed gradient with the sample layered as
38. 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 4 LR IM 24AC Using Tubes Bottles and Accessories 3 Filling and Plugging OptiSeal Tubes Filling the Tubes LR IM 24AC 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 NOTE If air bubbles occur in the tube shoulder area tilt and rotate the tube before it is completely filled 2 Afte a to wet the tube Homogeneous solutions of gradients and sample may be loaded into the tubes and centrifuged immediately See Gradient Preparation above 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 r filling the tube make sure that there is no fluid in the stem Draw off exce
39. the centrifuge by lifting it straight up and off the drive hub 2 Ifarotor vise is required set the rotor in the rotor vise 3 Remove the rotor plugs taking care to apply downward pressure on the plug adapter to avoid stripping the plugs 4 Remove spacers with the appropriate removal tool or a hemostat a Use removal tool 338765 to remove floating spacers Tube Removal Tool 361668 5 Remove tubes with the extraction tool 361668 6 Refer to CHAPTER 3 for sample recovery methods a LR IM 24AC 6 9 Using Vertical Tube and Near Vertical Tube Rotors Operation LR IM 24AC CHAPTER 7 Care and Maintenance _ Introduction This chapter 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 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
40. to eliminate unnecessary weight and minimize stresses LR IM 24AC 4 1 Using Fixed Angle Rotors Description Figure 4 1 Fixed Angle Rotors Axis of Rotation Area Axis of Rotation i l e ini EFi Tay ava max Axis of Rotation ____ ae 2nd Row 3rd Row Axis of Rotation 4 2 Type 100 Ti Type 70 1 Ti Type 50 4 Ti Type 25 LR IM 24AC Using Fixed Angle Rotors Description Table 4 1 General Specifications for Beckman Coulter Preparative Fixed Angle Rotors Relative Radial Distances mm Number of Maximum Centrifugal Tube Tubes x Tube Rotor Speed Field x g Angle Capacity Type rpm at Fmax degrees max lay Fmin k 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 8x 13 5 75 Ti 75 000 502 000 25 5 79 7 58 3 36 9 35 8x 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 46 8 64 10 x 38 5 50 4 Ti 50 000 312 000 20 111 5 96 2 80 8 33 44x65 50 3 Ti 50 000 223 000 20 79 5 64 2 48 9 49 18 x 6 5 50 2 Ti 50 000 302 000 24 107 9 81 2 54 4 69 12 x 39 50 Ti 50 000 22
41. tool kit 338841 is required 3 Using Tubes Bottles and Accessories Capping Tubes Table 3 3 Tube Cap Assemblies for Open Top Tubes in Fixed Angle Rotors O ring Tube Cap Hex Set or Assembly Nut Crown screw Insert Gasket Stem Tube Type Rotor Type 8 mm 5 16 in 303624 303379 303809 303730 303377 ucc 90 Ti 80 Ti 75 Ti 70 1 Ti 65 50 Ti 50 40 303658 303379 303810 303370 303377 UC 50 3 Ti 13 mm 1 2 in 303113 301870 307004 344672 307005 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 3050224 301870 307004 344672 302331 SS 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 346256 301870 307004 803543 302312 344672 346246 thinwall PP 90 Ti 80 Ti 75 Ti 70 1 Ti UC SS 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 301870 307006 338864 302312 301869 302266 SS 90 Ti 70 Ti 65 60 Ti 55 2 Ti 50 2 Ti 50 Ti 50 45 Ti 42 1 40 35 21 330860 301870 330774 803543 302312 858046 330788 thinwall PP 70 Ti 65 60 Ti 55 2 Ti UC 50 2 Ti 50 Ti 50 45 Ti 42 1 40 35 21 338907 301870 338911 338864 302312 878572 338910 thickwallPP 90 Ti 70 Ti 65 60 Ti PC 55
42. tube e 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 1 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 1 Release the button and pull the bottle out 5 Remove adapters using the appropriate removal tool 6 Refer to CHAPTER 3 for sample recovery methods IN LR IM 24AC 4 9 4 Using Fixed Angle Rotors Operation Figure 4 2 Removal Tools Used in Fixed Angle Rotors 4 10 LR IM 24AC CHAPTER 5 Using Swinging Bucket Rotors E Introduction This chapter 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 CHAPTER 2 for labware selection information and CHAPTER 3 for recommended filling and sealing or capping requirements and for sample recovery procedures Refer to CHAPTER 7 for information on the care of rotors and accessories Description Swing
43. tube Like the vertical tube rotors near vertical tube rotors use only Quick Seal and OptiSeal tubes Refer to CHAPTER 6 for specific information about the use of near vertical tube rotors Table 1 1 lists Beckman Coulter preparative rotors by use LR IM 24AC 1 5 Rotors Rotor Selection Table 1 1 Beckman Coulter Preparative Rotors by Use Relative Number of Nominal Maximum Centrifugal Tubes x Nominal Rotor For Use in Speed Field xg k Capacity mL Capacity Instruments Rotor rpm at Fmax Factor of Largest Tube mL Classified Rotors for Centrifuging Extremely Small Particles NVT 100 100 000 750 000 8 8x5 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 8x5 1 40 8 H R S Type 90 Ti 90 000 694 000 25 8x 13 5 108 H R S VTi 90 90 000 645 000 6 8x5 1 40 8 H R S Type 80 Ti 80 000 602 000 28 8 x 13 5 108 H R S VTi 80 80 000 510 000 8 8x 5 1 40 8 H R S Type 75Ti 75 000 502 000 35 8x 13 5 108 GIH RS NVT 65 2 65 000 416 000 16 16 x 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 8x 13 5 108 H R S VTi 65 65 000 404 000 10 8x5 1 40 8 H R S Type 65 65 000 368 000 45 8x 13 5 108 G9 H R S Type 50 Ti 50 000 226 000 78 12 x 13 5 162 G9 H R S Rotors for Centrifuging Small Particles in Volume Ty
44. which if not avoided will result in death or serious injury WARNING indicates a potentially hazardous situation which if not avoided could result in death or serious injury CAUTION indicates a potentially hazardous situation which if not avoided may result in minor or moderate injury IMPORTANT IMPORTANT is used for comments that add value to the step or procedure being performed Following the advice in the Important adds benefit to the performance of a piece of equipment or to a process NOTE NOTE is used to call attention to notable information that should be followed during installation use or servicing of this equipment 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 maintenance 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 centrifuge 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 centrifuge rotor tube system LR IM 24AC iii Safety Notice Chemical and Biological Safety Chemical and Biological Safety Normal operation may involve the use of solutions and t
45. 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 Drive Pins 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 acceleration Heavier rotors do not require the use of drive pins For swinging bucket rotors an indentation on Ss J the rotor adapter or the position of the mechanical overspeed cartridges see Overspeed Protection below indicates the location nei ees of the drive pins In this way the pins can be properly aligned m without lifting the rotor and dislocating the buckets Rotor Selection Selection of a rotor depends on a variety of conditions such as sample volume number of sample components to be separated particle siz
46. your laboratory safety officer Only the components and accessories listed in the applicable rotor manual should be used Fixed angle rotors can accommodate a variety of tube types listed in the rotor manual Refer to CHAPTER 3 for tube filling and sealing or capping requirements Observe the maximum rotor speeds and fill volumes listed in the applicable rotor instruction manual 4 3 4 Using Fixed Angle Rotors Rotor Preparation and Loading Fill volumes maximum rotor speeds and capping requirements for ultracentrifuge bottles are listed in CHAPTER 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 Prerun Safety Checks 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 2 Make sure the rotor is equipped with the correct overspeed disk refer to CHAPTER 1 If the disk is missing or damaged replace it as described in CHAPTER 7 eo 20 Sector a 355539 QB 3 Check the chemical compatibilities of all materials used Refer to APPEN
47. 0 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 LR IM 24AC XV Classification Program Introduction Instrument Classification All Model L2 65 s classified D 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 s 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 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 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 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
48. 0284 1 3356 4 41 1 0 244 1 377 1 3696 37 509 5 3 026 1 0365 1 3364 5 51 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 7
49. 2 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 G4H R S SW 60 Ti 60 000 485 000 45 6x4 24 G4H R S SW 55 Ti 55 000 368 000 48 6x5 30 G4H 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 6x 14 84 G4H R S SW 32 Ti 32 000 175 000 204 6 x 38 5 231 H R S SW 28 1h 28 000 150 000 276 6x17 102 C D F G H R S Rotors for Larger Volume Density Gradients SW 32 1 32 000 187 000 228 6x17 102 H R S LR IM 24AC 1 7 1 Rotors Rotor Selection Table 1 1 Beckman Coulter Preparative Rotors by Use Continued Relative Number of Nominal Maximum Centrifugal Tubes x Nominal Rotor For Use in Speed Field xg k Capacity mL Capacity Instruments Rotor rpm at Fmax Factor of Largest Tube mL Classified SW 30 1 30 000 124 000 138 6x8 48 B C D F G H R S SW 30 30 000 124 000 138 6x20 120 B C D F G H R S Rotors for Larger Volume Density Gradients continued SW 28h 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 3x 34 102 A B C D F G H Q R S a Rotors listed in parenthe
50. 3 os o on gt s cs Sox Sox aiso S E SN gt 8 82 5 58 3858 258 5X 25 a gt 55 ma fm S epee S eee S Ge aA polypropylene yes no yes yes yes yes yes yes no Ultra Clear no no yes yesi yes yes yes yes no polycarbonate yes no yes yesa no yesf yes yes no polypropylene yes no yes yes yes yes9 yesh yes no polyethylene no no yes yes yesi yes yes yes yes cellulose no no no no no yes yes yes no propionate stainless steel yes yes yes yes yesi no yes yes no a 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 i about specific solutions b 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 sam oan Below 26 C only i Below 21 C only j Marginal inky Crazing LR IM 24AC Cracking Do not use if there is methanol in the formula Tube life will be reduced by autoclaving Discoloration may occur Can be used if diluted Inspect containers and accessories before use nformation Inspect tubes and bottles for cracks or any major def
51. 3 13 3 Using Tubes Bottles and Accessories Capping Tubes e Fill 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 e For swinging bucket rotors fill to within 2 or 3 mm of the top of the tube e Fill 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 Stainless Steel 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 Capping Tubes Caps must be used with thinwall polypropylene and Ultra Clear tubes in fixed angle rotors To prevent spillage thickwall polypropylene polycarbonate 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 depe
52. 46 1 2858 1 3607 30 385 7 2 291 1 856 1 4137 63 1167 3 6 945 1 298 1 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 a Density data are from International Critical Tables b Computed from the relationship p25 10 2402 nD25 12 6483 for densities between 1 00 and 1 37 and p2 gt 10 8601 nD25 13 4974 for densities above 1 37 Bruner and Vinograd 1965 c Divide by 10 0 to obtain w v LR IM 24AC D 3 D Gradient Materials Description Table D 3 Density Refractive Index and Concentration Data Sucrose at 20 C Molecular Weight 342 34 Density Refractive by mg mL of Molarity Density Refractive by mg mL of Molarity g cm3 Index nD Weight Solution g cm3 Index nD Weight Solution 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
53. 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 45 541 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 a Density data are from International Critical Tables b Divide by 10 0 to
54. 6 000 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 10 x 10 45 Ti 45 000 235 000 24 103 8 69 8 35 9 133 6 x 94 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 mL 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 5004 25 132 1 122 8 113 4 62 100 x 1 21 21 000 60 000 18 121 5 90 9 60 3 402 10 x 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 a Rotors in parentheses are no longer manufactured b 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 c Maximum RCF measured at outer row d Maximum RCF measured at the third row Radial distances are those of the third row Tubes and Bottles LR IM 24AC 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
55. 60 Ti Rotor 120 5 20 Gradient Particle densities are 1 4 and 1 8 g mL 110 100 90 80 Distance Sedimented in Millimeters 70 0 0 2 0 4 0 6 0 8 Use of the w2t Integrator Reproducing Band Positions Refer to Figure B 1 pelusWIpes eouejsiq jeuooes4 2 4 2 8 3 2 3 6 4 0 pelusWIpes eoue siq jeuooes4 1 2 1 4 1 6 1 8 2 0 B Use of the w2t Integrator Calculating Sedimentation Coefficients 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 centrifugation time The value of wt 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 30 gradient at 20 C the value of s t is 0 92 from the figure By dividing the value of swt by the product wt from the integrator the result is the sedimentation coefficient in seconds of the particle pst B 5 LR IM 24AC APPENDIX C The Use of Cesium Chloride Curves _ Cesium Chloride Curves LR IM 24AC 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
56. 65 1 60 i 1 55 1 50 T D Z 145 D Ss A S es 1 40 v8 fS ay 1 35 H 1 30 _ _ N S LS 1 25 gy H H lo 1 20 r lt N a S igs 1 15 As f Z s LV S 1 10 aS SHAS S A ts amp SS 1 05 4 Y oo SP ay oo 1 00 34 2 46 1 55 2 63 0 76 5 Tmin Distance from Axis of Rotation mm Tmax Centrifugation of homogeneous CsCl solutions at the maximum allowable speeds from Figure C 1 results in gradients presented here Density increases from the top 34 2 mm to the bottom 76 5 mm of the tube LR IM 24AC C 3 The Use of Cesium Chloride Curves Typical Examples for Determining CsCl Run Parameters NOTE The curves in Figure C 1 and Figure C 2 are for solutions 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 CsCl 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 centrifuged 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
57. 7960 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 LR IM 24AC 3 29 Using Tubes Bottles and Accessories Making Ultra Clear Tubes Wettable Making Ultra Clear Tubes Wettable 3 30 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 Isopropanol 50 mL was slowly added to the hot solution and stirring and heating continued until a clear solution was obtained The solution was then allowed to cool to room temperature 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 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 Finally the tubes were briefly flushed with water tapped to remove excess liquid and left to dry LR IM 24AC CHAPTER 4 Using Fixed Angle Rotors _ Introduction This chapter contains ins
58. Communications 250 S Kraemer Blvd Brea CA 92821 U S A or are available at www beckmancoulter com A 1824 Plasmid Isolation Using NVT 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 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 For detailed information on a rotor see the applicable individual rotor manual LR IM 24AC E 1 References List of References E 2 A 2038 AR 8093 BA99 60495 BR 8101 BR 9272 DS 468 DS 514 DS 528 DS 694 DS 709 DS 728 DS 770 DS 793 DS 887 DS 9271 DS 9338 DS 9339 DS 9340 DS 9343 SR 171 SR 182 Preparation of Intestinal Mucins Using the NVT 65 Near Vertical Tube Rotor Fast Separations of Plasmid DNA Using Discontinuous Gradients in the Preparative Ultracentrifuge Rotor Safety Guide Warranty and Care Ultracentrifuge Rotors Tubes amp Accessories Catalog Optima L XP Techniques of Preparative Zonal and Continuous Flow Ultracentrifugation Ultracentrifuge Methods for Lipoprotein Research Use of the w2t Integrator 30 Minute 2 Step Purification of Plasma Membranes from Cultured Cells g Max System Short Pathlengths in High Force Fields Optimizing Centrifugal
59. DIX A 4 Verify that tubes bottles and accessories being used are listed in the appropriate rotor manual a Table 4 2 Maximum Run Speeds and Tube Volumes for Uncapped Tubes in Fixed Angle Rotors Nominal Part Number Maximum Maximum Capless Speed rpm Dimensions Volume mm Polycarbonate Polypropylene mL Polycarbonate Polypropylene Rotor Type 7x20 343775 343621 230 mL 42 000 42 000 42 2 Ti 7x20 342303 230 mL 42 000 42 2 Ti 4 4 LR IM 24AC Table 4 2 Maximum Run Speeds and Tube Volumes for Uncapped Tubes in Fixed Angle Rotors Continued Using Fixed Angle Rotors Rotor Preparation and Loading Nominal Part Number Maximum Maximum Capless Speed rpm Dimensions Volume mm Polycarbonate Polypropylene mL Polycarbonate Polypropylene Rotor Type 8x51 355657 1 45 000 50 4 Ti 25 000 25 11x89 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 16 x 64 355647 355646 6 5 50 000 50 000 50 8 50 000 30 000 80 Ti 75 Ti 70 1 Ti 50 Ti 40 000 30 000 40 16 x 76 355630 355640 7 5 50 000 30 000 65 50 Ti 8 55 000 30 000 90 Ti 16 5 45 000 20 000 70 Ti 60 Ti 55 2 Ti 50 2 Ti 25 x 89 355631 355642 24 40 000 20 000 42 1 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 38 x 102 355628 355643 44 30 000 15 000 45 Ti
60. 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 t Ithas been reported however that polypropylene tubes have been made wettable by soaking them in a chromic acid bath for about 30 minutes see Preparation of Polypropylene 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 chapter LR IM 24AC 3 1 Using Tubes Bottles and Accessories Gradient Preparation 1 mL Syringe ___ 20 to 22 Gauge Needle g S 2 to 3mm Gradient Buffer Sample with Gradient 2 to 3 Sucrose Added Cesium Chloride Gradients You can also prepare preformed step gradients by hand using a pipette Carefully layer solutions of decreasing concentration by placing the tip of the pipette at the angle formed by the tube wall and the meniscus or float the lighter gradient concentrations up by adding increased density solutions to the tube bottom using a hypodermic syringe with a long needle such as a pipetting needle Another way to forma linear gradient is to allow a step gradient to diffuse to linearity Depending on
61. Instructions For Use Rotors and Tubes For Beckman Coulter Preparative Ultracentrifuges LR IM 24AC February 2014 Beckman Coulter Inc 250 S Kraemer Blvd Ci aal Brea CA 92821 U S A OULTER Rotors and Tubes for Beckman Coulter Preparative Ultracentrifuges LR IM 24AC February 2014 2011 2014 Beckman Coulter Inc All rights reserved No part of this document may be reproduced or transmitted in any form or by any means electronic mechanical photocopying recording or otherwise without prior written permission from Beckman Coulter Inc Beckman Coulter Optima Quick Seal and the stylized logo are trademarks of Beckman Coulter Inc and are registered in the USPTO All other trademarks service marks products or services are trademarks or registered trademarks of their respective holders Find us on the World Wide Web at www beckmancoulter com Printed in U S A Safety Notice Read all product manuals and consult with Beckman Coulter trained personnel before attempting to operate instrument Do not attempt to perform any procedure before carefully reading all instructions Always follow product labeling and manufacturer s recommendations If in doubt as to how to proceed in any situation contact your Beckman Coulter Representative Alerts for Danger Warning Caution Important and Note DANGER DANGER indicates an imminently hazardous situation
62. Medic Co Ltd Tokyo Flammability hazard Do not use in or near operating ultracentrifuges LR IM 24AC Care and Maintenance Tube Cap Care Inspection Inspect tube cap components before each use Refer to Table 3 3 in CHAPTER 3 of this manual for a list of cap components e Carefully inspect the crown for deformed or roughened edges Run your finger around the bottom edge of the crown surfaces should be flat squared off and not rounded or jagged Check the top of 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 tools that have burrs A burred tool can score the crown Discard a damaged crown as it may fail and damage the rotor Inspect Here 2 Tube Cap Crown e 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 x SSZ S Tube Cap Stem e 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 e On caps with filling holes inspect the filling hole setscrew and threads If the hex cavity in the setscrew shows signs of wear replace the setscrew Nylon Insert Replacement Th
63. 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 e Ethanol 70 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 Inspection Frequent and thorough inspection is crucial to maintaining a rotor in good operating condition 1 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 a If any of these signs are evident do not run the rotor b Contact your Beckman Coulter representative for information about the Field Rotor Inspection Program and the Rotor Repair Program 2 Regularly check the condition of O rings or gaskets and replace any that are worn or damaged Handle O Ring SS Lid O Ring Threads Check for Corrosion Bs Overspeed Disk 3 Regularly check that all sealing surfaces are smooth and undamaged to ensure proper sealing Flammability hazard Do not use in or near operating ultracentrifuges 7 4 LR IM 24AC Care and Maintenance 7
64. Noryl plug 358275 64 x 124 Noryl 358977 356013 250 190 Type 16 16 000 c 32 x 124 355666 250 190 Type 16 14 000 334915 358326 64 x 124 Noryl 358977 356011 250 190 Type 16 16 000 a Bottles are polycarbonate unless otherwise indicated b 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 c Available only as bottle and cap assembly d Above 20 000 rpm insert assembly 355601 must be used e Polypropylene 3 24 LR IM 24AC Using Tubes Bottles and Accessories 3 Sample Recovery _ Black Cap bottles with three piece cap assemblies as follows Noryl Ca ii e 1 Be sure the O ring plug and bottle lip are dry and free of lubrication eee 2 Place the O ring on the underside of the plug Nory Plug ne Aluminum Cap 3 Insert the plug into the neck of the bottle ensuring ee that no fluid contacts the O ring O ring 4 Tighten the cap by hand Polycarbonate Bottle Sample Recovery If disassembly reveals evidence of leakage you should assume that some fluid escaped the container or rotor Apply appropriate decontamination procedures to the centrifuge rotor and accessories Sample recovery depends on the type of labware used the component s isolated and the analysis desired The Beckman Coulte
65. R 3 provides instructions for using tubes and related accessories CHAPTER 4 contains step by step procedures for preparing a fixed angle rotor for a centrifuge run Similar information for swinging bucket rotors is in CHAPTER 5 and CHAPTER 6 contains the same type of information for vertical tube and near vertical tube rotors Analytical continuous flow and zonal rotors are not covered in this manual CHAPTER 7 provides rotor tube bottle and accessory care and maintenance information as well as some diagnostic hints Please read it Proper rotor care results in longer rotor life 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 w2t integrator APPENDIX C describes the use of cesium chloride curves APPENDIX D contains reference information on some commonly used gradient materials APPENDIX E lists references for further reading Glossary provides a glossary of terms xiii Scope Scope of this Manual xiv LR IM 24AC Classification Program _ Introduction 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
66. Sealing Quick Seal Tubes Method B Without the Seal Guide NOTE Always apply the tip of the Tube Topper vertically 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 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 NOTE It is very important to apply the heat sink immediately 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 e 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 f The tube is now ready for centrifugation Seal the remaining tubes g Return the Tube Topper to its charging stand when finished 3 12 LR IM 24AC
67. a zone on top of the gradient RCF Relative centrifugal field the ratio of the centrifugal acceleration at a specified radius and speed rw 2 to the standard acceleration of gravity g according to the following equation RCF ro g where r is the radius in millimeters is the angular velocity in radians per second 27 RPM 60 and g is the standard acceleration of gravity 9807 mm s2 Thus the relationship between RCF and RPM is 2 Z M RCF 1 12r TM Fmax 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 min 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 LR IM 24AC Sedimentation 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 coefficient s Sedimentation velocity per unit of centrifugal force Silicone rubber A large group of silicone elastomers used in various accessories should be used at temperatures between 59 and 232 C 75 and 450 F Solution 555 Beckman 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 Spinkote Beck
68. ances for Beckman Coulter Centrifugation Products A 1 Use of the w2t Integrator B 1 w2t Integrator B 1 Reproducing Band Positions Refer to Figure B 1 B 2 Calculating Sedimentation Coefficients B 4 The Use of Cesium Chloride Curves C 1 Cesium Chloride Curves C 1 Typical Examples for Determining CsCl Run Parameters C 4 Gradient Materials D 1 Description D 1 References E 1 List of References E 1 Glossary Ultracentrifuge Rotor Warranty Ultracentrifuge Rotor Warranty Illustrations Illustrations 1 1 1 2 13 1 4 1 5 Sa 9 2 33 3 4 3 5 4 1 4 2 gl id 6 1 6 2 6 3 B 1 Gel C 2 Fixed Angle Swinging Bucket Vertical Tube and Near Verti cal Tube Rotors 1 2 Particle Separation in Fixed Angle Swinging Bucket Vertical Tube and Near Vertical Tube Rotors 1 4 Sedimentation Coefficients in Svedberg Units for Some Com mon Biological Materials 1 10 Nomogram 1 12 Arranging Tubes Symmetrically in a Rotor 1 15 Filling OptiSeal Tubes 3 7 The Cordless Quick Seal Tube Topper 3 9 Tools Used to Assemble Tube Caps 3 20 Tube Cap Installation 3 22 Tube Cap Vise 3 23 Fixed Angle Rotors 4 2 Removal Tools Used in Fixed Angle Rotors 4 10 Swinging Bucket Rotors 5 2 Checking Hook on Bucket Positions After the Rotor is Installed 5 9 Vertical Tube Rotors 6 3 Near Vertical Tube Rotors 6 3 Preparing a Vertical Tube or Near Vertical Tube Rotor 6 7 The sw2t Charts for the SW 60
69. arly 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 LR IM 24AC Rotors General Operating Information 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 manual When using dense solutions in plastic labware determine maximum run speed using the following square root reduction formula EQ8 reduced run speed maximum rated speed A e 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 publicati
70. at them individually ee ee O ring No fluid Appeals above O ring as wide black line Filling and Sealing Quick Seal Tubes 3 8 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 requirements 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 Seal Tubes with the Beckman Cordless Tube Topper 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 A sample application block 342694 is available for holding and compressing tubes and can be used to layer samples on preformed gradients in polypropylene Quick Seal tubes LR IM 24AC Using Tubes Bottles and Accessories 3 Filling and Sealing Quick Seal Tubes 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
71. be a thin flexible tube wall is required Ultra Clear or polypropylene tubes should be used in these cases depending on the need for transparency Labware Types NOTE Tubes made of cellulose nitrate were formerly used for various separations particularly rate zonal separations Beckman Coulter discontinued 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 Polypropylene Tubes Polypropylene tubes are translucent or transparent in appearance depending on wall thickness and are non wettable although some polypropylene tubes can be chemically treated to make them wettable Polypropylene tubes are reusable unless deformed during centrifugation or autoclaving Polypropylene tubes have good tolerance to gradient media including alkalines They are satisfactory for many acids bases alcohols DMSO and some 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 Several types of polypropylene tubes are available Open Top Polypropylene Tubes Thinwall open top tubes are used in swinging bucket and fixed angle rotors In z swinging bucket rotors thinwall tubes should be filled to within 2 or 3 mm o
72. ble 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 accelerates then seat against the rotor body for support 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 support for the hydrostatic forces generated by centrifugation Rotors General Description Materials Figure 1 1 Fixed Angle Swinging Bucket Vertical Tube and Near Vertical Tube Rotors Fixed Angle Rotor Swinging Bucket Rotor Vertical Tube Rotor Near Vertical Tube Rotor 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 c
73. ck 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 2 Regularly apply a thin uniform coat of Spinkote lubricant 306812 on the stem threads 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 2 and Table 3 4 for required tools and torque requirements 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 LR IM 24AC 3 Using Tubes Bottles and Accessories Capping Tubes Figure 3 3 Tools Used to Assemble Tube Caps Torque Wrench 858121 Nylon Insert me Tool 302460 Hex Driver ae 841883 Hex Driver a 841884 x Socket 858123 s Socket 870432 Removal Tool Ce 301875 S Socket Adapter 858122 Table 3 4 Required Tools and Torque Values 338906 blue Cap Nut Size Tightening Tool T
74. d the tube in place during plug removal 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 7 Extraction Tool Rack Insert 4 Use a slight twisting motion to slowly release any residual internal pressure when pulling the plug assembly from the tube 5 Repeat for each tube a 3 28 LR IM 24AC Quick Seal Tubes gt Cut Quick Seal stem here to provide an air inlet Sample out CentriTube Slicer 347960 Using Tubes Bottles and Accessories Sample Recovery There are several methods of recovering fractions from Quick Seal tubes One of the following procedures may be used NOTE If you plan to collect particles from the tube side or bottom first create an air passage by snipping the stem or inserting a hollow hypodermic needle in the top of the tube Puncture the side of the tube just below the 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 snipping off the tube stem then aspirating the sample with a Pasteur pipette or needle and syringe Slice the tube using the Beckman CentriTube Slicer 34
75. del ultracentrifuges classified other than G H R or S and some F have a mechanical overspeed system ote amp 75 000 rpm p 24 Sector a 334217 a Pant Drive Pin Cartridge Cartridge 1 16 All Beckman Coulter preparative rotors are shipped with an overspeeddisk 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 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 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 regul
76. e Zonal and Continuous Flow Ultracentrifugation publication DS 468 d 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 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 Rotor Preparation and Loading LR IM 24AC Swinging bucket rotors can accommodate a variety of tube types listed in the applicable rotor manual Refer to CHAPTER 3 for tube filling and sealing or capping requirements Observe the maximum rotor speeds and fill volumes listed in the rotor instruction manual For runs at other than room temperature refrigerate or warm the rotor beforehand for fast equilibration NOTE All buckets loaded or empty must be positioned on the rotor body for every run 5 3 5 Using Swinging Bucket Rotors Rotor Preparation and Loading Prerun Safety Checks Read all safety information in the rotor manual before using the rotor
77. e 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 m Fixed angle rotors are general purpose rotors that are f especially 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 in reduced run times Refer to CHAPTER 4 for specific information about the use of fixed angle rotors LR IM 24AC 1 3 Rotors Rotor Selection Figure 1 2 Particle Separation in Fixed Angle Swinging Bucket Vertical Tube and Near Vertical Tube Rotors At Speed At Rest in Rotor At Rest Outside Rotor Fixed Angle Rotors Pathlength Swinging Bucket Rotors Pathlength Vertical Tube Rotors Imin max c Pathlength Near Vertical Tube Rotors r min n max Pathlength Dark gray represents pelleted material light gray is floating components and bands are indicated by black lines 1 4 LR IM 24AC Rotors 1 Rotor Selection Swinging bucket rotor are used fo
78. e t Conversely if the value of w t is known sedimentation coefficients can be estimated from zone positions Refer to APPENDIX B of this manual for an explanation of the swt 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 lt 5 to 20 or if fixed angle vertical tube or near vertical tube 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 reorientation process General Operating Information 1 14 Careful centrifugation technique is essential because forces generated 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 Classification Progra
79. e must be correctly mounted with the clamping knob positioned on the right or crimping of the crown may result LR IM 24AC 3 23 Using Tubes Bottles and Accessories Filling and Capping Tubes Table 3 5 Available Bottles Assembly and Operation Volume Bottle Required Cap Assembly mL Bottle and Maximum Part Dimensions Part Cap Max Min speed Required Number mm Material Number Assembly Rotor rpm Adapter 355656 16x 64 Noryl 355604 355615 8 5 8 5 Type 50 50 000 355651 16x76 Noryl 355604 355603 10 4 5b Types 90Ti 65 000 80 Ti 75 Ti 70 1 Ti 65 Type 50 Ti 50 000 Type 40 40 000 c 11x79 polypropylene 355672 10 10 Type 28 20 000 342327 870329 355654 25x89 aluminum 355619 355618 26 3 165 Types 70 Ti 60 000 60 Ti Type 55 2 Ti 55 000 Type 50 2 Ti 50 000 Type 42 1 42 000 Noryl 355617 355616 26 3 16 Type 30 30 000 355670 29x 102 polypropylene 355601 357001 50 40 Type 28 20 0009 Type 16 14 000 356977 c 29 x 102 polypropylene 355601 3570008 50 40 Type 28 20 0009 Type 16 14 000 356977 c 29 x 102 357003 50 40 Type 16 14 000 356977 Type 28 20 000 355655 38 x 102 aluminum 355623 355622 70 355 Type 45 Ti 45 000 Noryl 355621 355620 70 35 Type 35 35 000 Type 21 21 000 c 52 x 134 355674 150 150 Type 16 5 000 339362 355627 60x 120 Delrin 362247 334025 250 250 Type 19 19 000 w
80. e 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 Setscrew LR IM 24AC 7 13 7 Care and Maintenance Returning a Rotor or Accessory to the Factory 2 Unscrew the insert 3 Fitanewinsert 302312 on the end of the tool and screw it into the stem until it bottoms firmly against the stem threads N Returning a Rotor or Accessory to the Factory Before returning a rotor or accessory for any reason prior permission must be obtained from Beckman Coulter Inc This form may be obtained from your local Beckman Coulter sales office The form entitled Returned Material Authorization RMA for United States returns or Returned Goods Authorization RGA for international returns should contain the following information e rotor type and serial number e history of use approximate frequency of use e reason for the return e original purchase order number billing number and shipping number if possible name and email address of the person to be notified upon receipt of the rotor or accessory at the factory and e name and email address of the person to be notified about repair costs etc To protect our personnel it is the customer s responsibility to ensure that the parts are free from pathogens chemica
81. e 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 Many rotors can accommodate a variety of tube sizes by using adapters that line the tube cavity or bucket e Small open top tubes use Delrin adapters which line the tube cavity or bucket e 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 CHAPTER 1 In vertical tube rotors rin is unchanged see the illustration in F
82. each rotor 5 Use the required spacers and or floating spacers if necessary to complete the loading operation a If OptiSeal tubes are being used install a spacer over each plugged tube refer to the applicable rotor manual 1 Leave buckets without tubes completely empty Spacer a _ 7 Tube Plug _ Tube 5 5 5 Using Swinging Bucket Rotors Rotor Preparation and Loading b If Quick Seal tubes are being used install spacers and or floating spacers over sealed tubes refer to the applicable rotor manual e 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 1 Leave buckets without tubes completely empty g Max Metal Floating Spacer Spacer lil Y Dome Top Bell Top 6 Match numbered caps with numbered buckets a Screw the caps into the bucket until there is metal to metal contact b Tighten flat caps with a screwdriver NOTE For SW 32 Ti and SW 32 1 Ti rotors use a lint free cotton swab to apply Spinkote lubricant 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 clockwise until it stops one quarter turn 7 Attach all buckets loaded or empty to the rotor Loaded buckets must be arranged symmetrically on the rotor see Figure 1 5
83. eckman Coulter Preparative Swinging Bucket Rotors Radial Distances mm k Factors Number of Maxi Relative Tubes x mum Centrifugal Tube Speed Field x g k g mL g mL g mL Capacity Rotor rpm at Tmax Tmax Fav 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 60 000 485 000 120 3 91 7 63 1 45 126 115 111 6x4 SW 55 Ti 55 000 368 000 108 5 84 6 60 8 48 135 123 118 6x5 SW 50 1 50 000 300 000 107 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 40 000 285 000 158 8 112 7 66 7 137 368 338 325 6x 14 SW 32 Ti 32 000 175 000 152 5 109 7 66 8 204 468 428 412 6 x 38 5 SW 32 1 Ti 32 000 187 000 162 8 113 6 64 4 228 613 560 536 6x17 SW 30 1 30 000 124 000 123 0 99 2 75 3 138 393 360 346 6x8 SW 30 30 000 124 000 123 0 99 2 75 3 138 393 360 346 6 x 20 sw 28 14 28 000 150 000 1713 122 1 729 276 757 694 668 6x17 SW 284 28 000 141 000 161 0 118 2 75 3 246 680 622 600 6 x 38 5 SW 25 1 25 000 90 400 129 2 92 7 56 2 337 917 840 809 3x 34 a Rotors listed in parentheses are no longer manufactured b Maximum speeds are based on a solution density of 1 2 g mL in all swinging bucket rotors c Calculated for 5 to 20 wt wt sucrose at 5 C using the tables in Appendix of Techniques of Preparativ
84. elleting efficiency In Tmax Tmin 10 o 3600 or _ 253303 x In Tmax min k RPM 1000 2 LR IM 24AC Glossary Clearing time t t k s where tis time in hours k is the clearing factor of the rotor and s is the sedimentation coefficient in Svedberg units S CsCl Cesium chloride a high density salt used in solution in isopycnic separations to separate particles based on their density CsSO Cesium sulfate a salt similar to CsCl that will form its own gradient in solution Delrin Thermoplastic material acetal homopolymer used for most tube adapters Delrin is a registered trademark of E I Du Pont de Nemours amp Company Density Mass per unit volume Density separation A centrifugal separation process based on differences in particle densities Differential separation A centrifugal separation process based on differences in particle sizes EPDM Ethylene propylene rubber used for O rings and pad adapters should be used at temperatures between 57 and 120 C 70 and 250 F Ethidium bromide A fluorescent intercalating orange dye used commonly in the separation of DNA and in gel electrophoresis Fixed angle rotor A rotor in which the tubes are held at an angle usually 20 to 45 degrees from the axis of rotation g Max A system of centrifugation using a combination of short Quick Seal tubes and floating spacers designed to reduce volume
85. en the tube is held upright there will be about 3 2 mm of interband separation NOTE In swinging bucket rotors the interband separation after centrifugation is the same as during centrifugation as there is no gradient reorientation 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 do up cos 0 where d is the interband separation achieved during centrifugation and 0 is the tube angle Example B Knowing particle densities 1 50 and 1 52 g mL how do you achieve good separation 1 In Figure C 2 sketch ina horizontal line corresponding to each particle s buoyant density 2 select the curve at the desired temperature 4 C and tube volume full that gives good separation 3 Note the speed indicated along the curve 50 000 rpm 4 From Figure C 1 determine the maximum allowable homogeneous 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 rr h where r is the tube radiu
86. er detergents and temperatures 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 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 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 polypropylene tubes are intended for one time use and should be discarded after use Decontamination eo 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 LR IM 24AC 7 7 Care and Maintenance Tube Bottle and Accessory Care Sterilization and Disinfection Guo uy 7 8 121 C Refer to Table 7 1 for sterilization methods recommended for each container type Most tubes and accessories except those made of Ultra Clear p
87. ertical 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 Rotors 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 8 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 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 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 user s sample non Beckman Coulter tubes adapters or other rotor contents This warranty is void if the Beckman Coulter Centrifuge System 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 Thi
88. ertical Tube Rotors 6 Rotor Preparation and Loading 6 Tighten each rotor plug as shown in Figure 6 3 Refer to Table 6 2 for the correct tightening tools and torque values a To avoid stripping the plugs apply downward pressure to the plug adapter while tightening the plugs e 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 b Make sure that all plugs are level with each other 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 Remove the rotor from the vise Figure 6 3 Preparing a Vertical Tube or Near Vertical Tube Rotor Assembly Plug N Potor Plug Gasket See Table 6 2 for the correct tightening tools and torque values LR IM 24AC 6 7 Using Vertical Tube and Near Vertical Tube Rotors Operation Operation For runs at other than room temperature refrigerate or warm the rotor beforehand for fast equilibration 1 Carefully lower the rotor straight down onto the drive hub e Careful installation will prevent disturbing the sample or tripping the imbalance detector Lower the rotor straight down onto the drive hub 2 Refer to the centrifuge instruction manual for detailed operating information N
89. es and or long run times The relative pelleting efficiency of each rotor is measured by its k factor clearing factor EQ 1 ka In Tmax Tmin T 10 o 3600 where is the angular velocity of the rotor in radians per second 2nRPM 60 or 0 10472 x rpm Tmax is the maximum radius and r min is the minimum radius 1 8 LR IM 24AC LR IM 24AC Rotors Rotor Selection After substitution EQ2 p 2533 x 1015 In imax tnin rpm This factor can be used in the following equation to estimate the time t in hours required for pelleting EQ3 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 Sis equal to 10713 seconds It is usual practice to use the standard sedimentation coefficient 520 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 EQ4 k sd speed of a k actual run speed adj Run times can also be calculated from data established in prior experiments when the k factor of the previous rotor is known For any two rotors a and b EQ5 where the k factors have been adjusted for the actual run speed used Figure 1 3 lists sedimentation coefficients for some common biological materials The k factors at maximum speeds for Beckman Coulter prepa
90. est 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 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 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 the rotor 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 Dispose of all waste solutions according to appropriate environmental health and safety guidelines If disassembly reveals evidence of leakage you should assume that some fluid escaped the container or rotor Apply appropriate decontamination procedures to the centrifuge rotor and accessories Mechanical Safety Use only the rot
91. ethods a LR IM 24AC CHAPTER 6 Using Vertical Tube and Near Vertical Tube Rotors _ Introduction This chapter 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 CHAPTER 2 for labware selection information and CHAPTER 3 for recommended filling and sealing or capping requirements and for sample recovery procedures Refer to CHAPTER 7 for information on the care of rotors and accessories Description 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 Vertical Tube Rotors 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 LR IM 24AC 6 1 Using Vertical Tube and Near Vertical Tube Rotors Description Table 6 1 General Specifications for Beckman Coulter Preparative Vertical Tube and Nea
92. f 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 re orients during centrifugation the maximum partial fill volume depends on the tube angle For greater fill volumes use tubes with caps Refer P to the applicable rotor manual for fill volumes and speed reduction requirements Thickwall polypropylene tubes are typically reusable unless deformed during centrifugation or autoclaving LR IM 24AC 2 3 Tubes Bottles and Accessories Labware Types OptiSeal Tubes aa LY _ Spacer WE Plug 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 centrifugation 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 Quick Seal Polypropyle
93. for the Type 90 Ti rotor only Similar 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 dr 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 However 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 reduction 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 o
94. gle 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 transparent and designed for onetime 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 Stainless Steel Tubes LR IM 24AC 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 centrifuged 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 2 5 2 Tubes Bottles and Accessories Temperature Limits konical Tubes konical tubes used with conical adapters in swinging bucket rotors to optimize pelleting separatio
95. held Tube Topper Use the applicable tube rack listed in the appropriate rotor manual Figure 3 2 The Cordless Quick Seal Tube Topper Charging Stand 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 a Make sure the pushbutton is turned to LOCK position b Insert the ends of the Tube Topper tip into the two openings of the copper strips at the end of the Tube Topper device 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 LR IM 24AC 3 9 Using Tubes Bottles and Accessories Filling and Sealing Quick Seal Tubes 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 Seal Former 3 Seal each tube using Method A with the seal guide or B without the seal guide described below Method A is preferable when sealing smaller tubes or when resealing a tube that leaks 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
96. her if applicable and the nut e The nylon insert should already be installed in the stem a For titanium caps turn the crown slightly to be sure it is properly seated on the stem 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 LR IM 24AC 3 21 3 Using Tubes Bottles and Accessories Capping Tubes 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 e 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 a Tighten the nut by hand just enough to hold the tube cap in place Figure 3 4 Tube Cap Installation CORRECT WRONG O ring Tube beyond hs below the O ring e O ring resting on the crown LF A 4 Position the capped tube in the appropriate sized hole from the underside of the tube cap vise 305075 e 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 a 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 Use a syringe to finish filling the tube thro
97. ibosomes Poliomyelitis 200 Tobacco mosaic Equine encephalitis Polysomes Viruses Rous sarcoma 800 Feline leukemia Bacteriophage T2 Microsomes Subcellular Particles Plasma membranes Mitochondria 100 000 LR IM 24AC Rotors 1 Rotor Selection Isopycnic Separations LR IM 24AC A sedimentation equilibrium or isopycnic method separates particles 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 EQ6 d p m Pp Pyg 18u Rotors Rotor Selection Figure 1 4 Nomogram 100 000 95 000 90000 85000 80 000 60 000 1 000 000 900 000 800 000 700 000 600 000 500 000 50 000 400 000 300 000 200 000 100 000 90 000 C 80 000 4 sF 70 000 4 60 000 50 000 30 000 40 000 30 000 Wm 20 000 e 20 000 10000 9000 4 F 8000 4 7000 4 6 000 20 5000 4 000 ae 3 000 2000 aie 10 1000 E 10 000 Radial Distance Relative Centrifugal Rotor Speed mm Field x g rpm Align a straightedge through known values
98. ics and Chemical Resistances of Tube and Bottle Materials v vy G z B 5 v A E 2 g a 2 a z z x E ela g oO E 2 3 3 2 S S gt O 2 2 5 o w v 2 2 lt c A v o 5 F v D E 5 T v cS T T D 5 ro T a lt ma i S Q Dla n re lt N o YZ 5 O v 4 4 lt a o T o o z m A T e le le le E E amp a ja ja a a im wu IZ x O Ia thinwall transparent yes yes no S U U M S U U U U U S polypropylene thickwall translucent no no gt yes S S S M S M M U M U S polypropylene Ultra Clear transparent yes yes no M polycarbonate transparent no no yes M M U U M polypropylene translucent no nop yes S M S M M S transparent polyethylene transparent yes no yes S S S S S S U M M M S translucent cellulose transparent no nob 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 marginal resistance U unsatisfactory resistance a Refer to Appendix A for information about specific solutions b Polypropylene 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 NOTE This information has been consolidated from a number of sources and is provided only as a guide to the selection of tube
99. id 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 LR IM 24AC Tubes Bottles and Accessories Labware Types Polypropylene Tubes 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 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 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 without caps in fixed angle rotors Ultra Clear Tubes Ultra Clear tubes made of a tough thermoplastic are thinwall and not wettable but can be made wettable see CHAPTER 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 an
100. igure 1 2 However in fixed angle and near vertical tube rotors r min must be calculated LR IM 24AC Delrin is a registered trademark of E Du Pont de Nemours amp Company 2 7 2 Tubes Bottles and Accessories Adapters 2 8 Table 2 2 Quick Seal Tube Spacers Part Number Spacer Description 342883 black anodized aluminum 342418 clear anodized aluminum 342696 clear anodized aluminum 342695 red anodized aluminum 342699 red anodized aluminum 342417 clear anodized aluminum 342697 titanium p s EE 344389 white Delrin 344634 white Delrin 344635 344676 black Noryl 345828 black Noryl Dai 349289 blue anodized aluminum 358164 black Delrin Sz LR IM 24AC Tubes Bottles and Accessories Adapters EQ 9 Lmin Tna da sin cos 0 L sind where Tmax the distance in millimeters from the axis of rotation to the farthest part of the tube cavity d diameter of the tube in millimeters L 9 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 max and r min for a given rotor with a Delrin adapter Table 2 3 lists adapter dimensions used in the equation
101. ing 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 CII gt 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 anda 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 LR IM 24AC 5 1 Using Swinging Bucket Rotors Description Figure 5 1 Swinging Bucket Rotors Axis of Rotation SW 60 Ti Axis of Rotation SW 40 Ti Axis of Rotation Axis of Rotation 5 2 LR IM 24AC Using Swinging Bucket Rotors Rotor Preparation and Loading Table 5 1 General Specifications for B
102. ith the spacer a Separate the tube from the spacer with a twisting motion Spacer Removal Tool 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 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 361668 3 26 LR IM 24AC Using Tubes Bottles and Accessories 3 Sample Recovery 3 Place the tubes back into the tube rack e Openings in the rack allow the tubes to be pierced either from the bottom or sides permitting fractions to be easily collected regardless
103. l hazards and or radioactivity 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 RMA RGA form when mailing the rotor and or accessories Customers located outside the United States should contact their local Beckman Coulter office Diagnostic Hints Some of the more common operating problems experienced in centrifugation are listed Table 7 2 with suggestions for their solutions Contact Beckman Coulter Field Service if a problem cannot be corrected NOTE Use only the labware listed in the applicable rotor manual 7 14 LR IM 24AC Table 7 2 Troubleshooting Chart Care and Maintenance Diagnostic Hints Symptom Possible Cause and Suggested Action Rotors Severe vibration e 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 il
104. lace 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 e Insufficient liquid in tube Observe minimum fill volumes Tubes with snap on caps Tube too full the meniscus must be kept lower to prevent leakage Uncapped tubes Tube volume exceeds maximum uncapped volume Refer to the rotor manual for tube volumes and speed reductions OptiSeal tubes 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 Quick Seal tubes LR IM 24AC 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 7 Care and Maintenance Diagnostic Hints Table 7 2 Troubleshooting Chart Continued Symptom Possible Cause and Suggested Action Tube cracking e 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 centrifugation e Tubes may become brittle with age and use Dispose of brittle or cracked tubes
105. lene Tubes 2 5 vii Contents viii CHAPTER 3 Ultra Clear Tubes 2 5 Cellulose Propionate Tubes 2 5 Stainless Steel Tubes 2 5 konical Tubes 2 6 Bottles 2 6 Temperature Limits 2 6 Spacers and Floating Spacers 2 7 Adapters 2 7 Using Tubes Bottles and Accessories 3 1 Introduction 3 1 Gradient Preparation 3 1 Cesium Chloride Gradients 3 2 General Filling and Sealing or Capping Requirements 3 3 Filling and Plugging OptiSeal Tubes 3 4 Filling the Tubes 3 5 Seating the Tube Plugs 3 7 Filling and Sealing Quick Seal Tubes 3 8 Method A With the Seal Guide 3 11 Method B Without the Seal Guide 3 12 Filling Open Top Tubes 3 13 Open Top Polypropylene Tubes 3 13 Swinging Bucket Rotors 3 13 Fixed Angle Rotors 3 13 Other Open Top Tubes 3 13 Polycarbonate 3 13 UltraClear 3 13 Polypropylene 3 14 Polyethylene 3 14 Stainless Steel 3 14 Capping Tubes 3 14 Tube Cap Assemblies 3 15 Titanium Caps 3 15 Aluminum Caps 3 17 Inspecting and Lubricating Tube Caps 3 19 Assembling Tube Caps 3 19 Filling and Capping Tubes 3 23 Sample Recovery 3 25 Capped Tubes 3 25 OptiSeal Tubes 3 26 Removing Plugs from Tubes 3 28 Quick Seal Tubes 3 29 Making Ultra Clear Tubes Wettable 3 30 CHAPTER 4 CHAPTER 5 CHAPTER 6 CHAPTER 7 Contents Using Fixed Angle Rotors 4 1 Introduction 4 1 Description 4 1 Tubes and Bottles 4 3 Rotor Preparation and Loading 4 4 Preru
106. les 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 of the particle of interest the distance from the axis of rotation it is to travel its density and certain properties of the gradient For example to position a protein sample characterized by s of 7 x 10 13 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 swt must be 0 92 from the figure gpa B 4 7x10 1 31 x 10 rad s ot This value can be set into the integrator and the integrator used to terminate the run when this value is reached Because of deceleration however this value of wt 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 wt that accumulates during deceleration from run speed then subtract that value from the total determined from the charts The radial distance to the tube meniscus in the SW 60 Ti rotor is 63 mm B 2 LR IM 24AC LR IM 24AC Figure B 1 The swt Charts for the SW 60 Ti Rotor 20 C SW 60 Ti Rotor 120 10 30 Gradient Particle densities are 1 4 and 1 8 g mL 110 100 90 80 Distance Sedimented in Millimeters 70 0 0 4 0 8 1 2 1 6 SW
107. 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 LR IM 24AC 75 Care and Maintenance Rotor Care Overspeed Disk Replacement 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 Centering Tool 331325 S 4 Peel the paper backing off the new disk but do not touch the adhesive a Fit it adhesive side down around the centering tool b Press the disk firmly to the rotor bottom 5 Remove the tool a Allow the disk to set for a minimum of 2 hours 7 6 LR IM 24AC Care and Maintenance 7 Tube Bottle and Accessory Care Tube Bottle and Accessory Care Proper care of tubes and bottles involves observing temperature fill volume and run speed limitations as well as careful cleaning and sterilization procedures Cleaning Do not wash tubes and bottles in a commercial dishwash
108. lustrated in CHAPTER 1 Figure 1 5 e 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 CHAPTER 5 Stripped rotor plugs on vertical tube or near vertical tube rotors Rotor vise not used wrong tool used incorrect torque or insufficient pressure on plug adapter when tightening rotor plugs Observe careful tightening procedures Rotor lid is difficult to remove after centrifugation 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 Paint coming off where bucket contacts rotor pocket on swinging bucket rotor Not an operational problem Tubes Tube leakage Tubes with cap assemblies Caps not properly secured Caps must be properly seated on tubes and then fully tightened e Cap components not dry before assembly Thoroughly dry all components before assembling e 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 rep
109. m chart at the beginning of this manual and Table 1 1 Acceptable classification letters are engraved on rotor lids handles stands or bodies NOTE Specific information about filling sealing and capping containers loading rotors etc can be found in later sections LR IM 24AC Rotors General Operating Information 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 illustrated in Figure 1 5 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 Figure 1 5 Arranging Tubes Symmetrically in a Rotor O O O 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 Load the opposite rotor cavities or buckets with tubes containing a liquid of the same density as opposing tubes Use smaller tubes with adapters or smaller Quick Seal tubes with floating spacers to distribu
110. m fill volume includes sample and gradient Refer to individual rotor manuals for specific filling and capping requirements Table 3 1 Filling and Capping Requirements for Tubes and Bottles Tubes or Bottles Filling Level Requirements Swinging Bucket Rotors Fixed Angle Rotors Vertical and Near Vertical Tube Rotors Polypropylene thinwall tubes thickwall tubes OptiSeal tubes Quick Seal tubes konical Quick Seal tubes konical open top tubes within 2 3 mm of top at least 1 2 full full or 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 full and plugged full and heat sealed Quick Seal tubes full and heat sealed bottles min to max with screw on cap or cap assembly Table 3 3 Ultra Clear open top tubes within 2 3 mm of top full with cap full and heat sealed Polycarbonate thickwall tubes thickwall bottles at least 1 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 and bottles at least 1 2 full 1 2 to max capless level or full with cap or cap assembly tubes any level any level with cap or cap assembly Table 3 3 Cellulose Propionate tubes full 1 2 to max capless level no
111. man Coulter lubricant for metal to metal contacts Sucrose A sugar not a self forming gradient used in rate zonal separations generally used in separating RNA subcellular organelles and cell membranes Supernatant The liquid above the sedimented material following centrifugation Svedberg unit S A unit of sedimentation velocity 15 10 13 seconds Swinging bucket rotor A rotor in which the tubes or bottles are carried in buckets microtiter plate carriers or racks that swing up to the horizontal position during centrifugation sometimes referred to as a horizontal or swing out rotor Ultem Polyetherimide PEI used in adapters covers and spacers should be used at temperatures between 29 and 204 C 20 and 400 C Ultem is a registered trademark of GE Plastics Vertical tube rotor A rotor in which the tubes or bottles are held parallel to the axis of rotation Glossary Viton Fluorocarbon elastomer used in high temperature applications Viton is a registered trademark of E l Du Pont de Nemours amp Company Wettable Tube or bottle material that water or other aqueous solution will adhere to the more wettable a tube or bottle material is the more biological material DNA protein cells and so forth will adhere to the walls Glossary 3 Glossary Glossary 4 LR IM 24AC LR IM 24AC Ultracentrifuge Rotor Warranty All Beckman Coulter ultracentrifuge Fixed Angle V
112. n Safety Checks 4 4 Rotor Preparation and Loading 4 5 Operation 4 7 Installing the Rotor 4 7 Removal and Sample Recovery 4 8 Using Swinging Bucket Rotors 5 1 Introduction 5 1 Description 5 1 Tubes and Bottles 5 3 Rotor Preparation and Loading 5 3 Prerun Safety Checks 5 4 Rotor Preparation and Loading 5 4 Operation 5 7 Removal and Sample Recovery 5 9 Using Vertical Tube and Near Vertical Tube Rotors 6 1 Introduction 6 1 Description 6 1 Vertical Tube Rotors 6 1 Near Vertical Tube Rotors 6 2 Tubes and Bottles 6 4 Rotor Preparation and Loading 6 4 Prerun Safety Checks 6 4 Rotor Preparation and Loading 6 5 Operation 6 8 Removal and Sample Recovery 6 9 Care and Maintenance 7 1 Introduction 7 1 Rotor Care 7 1 Decontamination 7 3 Sterilization and Disinfection 7 3 Inspection 7 4 Field Rotor Inspection Plan 7 5 Lubrication 7 5 Contents APPENDIX A APPENDIX B APPENDIX C APPENDIX D APPENDIX E Overspeed Disk Replacement 7 6 Tube Bottle and Accessory Care 7 7 Cleaning 7 7 Decontamination 7 7 Sterilization and Disinfection 7 8 Tube and Bottle Storage 7 10 Removing Jammed or Collapsed Tubes 7 10 Tube Cap Care 7 10 Cleaning 7 11 Decontamination 7 11 Sterilization and Disinfection 7 12 Lubrication 7 12 Inspection 7 13 Nylon Insert Replacement 7 13 Returning a Rotor or Accessory to the Factory 7 14 Diagnostic Hints 7 14 Chemical Resist
113. nction 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 WARRANTY SHALL BE IN LIEU OF ALL WARRANTIES OF FITNESS AND OF THE WARRANTY OF MERCHANTABILITY AND BECKMAN COULTER INC SHALL HAVE NO LIABILITY FOR SPECIAL OR CONSEQUENTIAL DAMAGES OF ANY KIND WHATSOEVER ARISING OUT OF THE MANUFACTURE 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 Indianapolis Indiana 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 contact 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 ACCESSORIES ARE NON RADIOACTIVE NON PATHOGENIC NON TOXIC AND OTHERWISE SAFE TO SHIP AND HANDLE IS
114. nd Loading For runs 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 rotor 1 Make sure that the rotor plugs gaskets and spacers are clean and show no signs of corrosion or cracking e The high forces generated in these rotors can cause damaged components to fail 2 Make sure the rotor is equipped with the correct overspeed disk refer to CHAPTER 1 If the disk is missing or damaged replace it as described in CHAPTER 7 t s 20 Sector gt 355539 LJ nb 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 a 6 4 LR IM 24AC Using Vertical Tube and Near Vertical Tube Rotors 6 Rotor Preparation and Loading Rotor Preparation and Loading 1 Be sure that plug threads are clean and lightly but evenly lubricated with Spinkote lubricant 306812 Gasket 7 Threads C 2 Ifusing a rotor vise set the rotor into the vise which should be bolted or clamped to a rigid surface 3 Dry the exterior of the plugged OptiSeal or sealed Quick Seal tubes e Moisture between the tube and the rotor cavity may lead to tube collapse and increase the force required to extract the tube a Slide the tubes into the tube cavities e Tubes must be
115. nd 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 coefficients 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 20 wt wt linear sucrose gradients at 5 C These are called k factor and are given in Table 5 1 in CHAPTER 5 These constants can be used to estimate the time t in hours required to move a zone of particles of known sedimentation coefficient and density to the bottom of a5 to 20 gradient EQ7 a LR IM 24AC 1 13 Rotors General Operating Information 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 swt charts available in Use of the w t Integrator publication DS 528 If the values of s and w are known and gradients are either 5 to 20 or 10 to 30 wt wt sucrose you can use the charts to calculate the run tim
116. nd on the tube or bottle material diameter and wall thickness as well as on the rotor The applicable rotor instruction manual specifies which cap should be used with a particular tube or bottle 3 14 LR IM 24AC Using Tubes Bottles and Accessories Capping Tubes Tube Cap Assemblies gorg S iscisi A tube cap assembly includes O00 e oii a stem e anylon insert Delrin Washer an O ring or flat gasket Crown acrown a Delrin crown washer in red blue and black aluminum caps O ri ED Gasket r Ge ahex shaped nut and aa a stainless steel setscrew Slem The stem supports the upper portion of the tube To provide tube LR IM 24AC ae Th support during centrifugation the stem is longer for thinwall tubes than for thickwall or metal tubes Some stems have an abraded Tube 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 tightened and filled the setscrew is used to seal the filling hole in the stem by seating again
117. ne Tubes Metal Spacer AS Ly g Max Floating Spacer Dome Top Bell Top Polycarbonate Tubes 2 4 iS 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 e 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 appearance 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 Although 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 flu
118. ne quarter filled Also 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 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 approximate 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 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 C 1 The Use of Cesium Chloride Curves Cesium Chloride Curves Figure C 1 Precipitation Curves for the Type 90 Ti Rotor
119. ns 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 polypropylene 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 polypropylene 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 Type 16 and Type 28 rotors no longer manufactured use capped polypropylene bottles in addition to polycarbonate bottles The Type 19 rotor uses a polypropylene bottle with a 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 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
120. ntrifuge instruction manual for detailed operating information Lower the rotor straight down onto the drive hub Removal and Sample Recovery 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 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 3 Remove spacers and or floating spacers with a removal tool 338765 or hemostat 4 8 LR IM 24AC Using Fixed Angle Rotors Operation 4 Remove tubes or bottles from the rotor using one of the following procedures Refer to Figure 4 2 for removal tools 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 Extract capped tubes using the appropriate removal tool 1 Insert the threaded end of the tool into the cap and screw at least one turn e Ifnecessary turn the tube slightly to break any vacuum seal created between the tube and the cavity and pull the tube out 2 Use the hex key end of the removal tool to remove the cap setscrew but try not to squeeze the
121. obtain w v D 4 LR IM 24AC Gradient Materials Description Table D 4 Density Conversion for Cesium and Rubidium Salts at 20 C w w CsCl CsBr Csl Cs SO CsNO3 RbCl RbBr Rbi Rb2S04 RbNO3 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 21817 1 21980 1 2309 1 1952 26 1 24093 1 24990 1 24942 1 2643 1 22421 1
122. of the type of separation NOTE If you want 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 4 Use one of the following methods to retrieve the sample a Puncture the side of the tube just below the sample band with a needle and syringe and draw the sample off e Take care when piercing the tube to avoid pushing the needle out the opposite side KEI b Puncture the bottom of the tube and collect the drops Bs Sample out c 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 P d Slice the tube using the Beckman CentriTube Slicer 303811 e Refer to publication L TB 010 for instructions for using the CentriTube Slicer LR IM 24AC 3 27 Using Tubes Bottles and Accessories Sample Recovery 1 Use CentriTube Slicer 347960 and CentriTube Slicer Adapter 354526 for 13 mm tubes e Tubes are pressurized after centrifugation so pierce the tube top with a needle to relieve pressure before slicing ny t CentriTube Slicer 347960 Removing Plugs from Tubes 1 Place the tube rack insert over the tubes in the rack 2 Press down on the rack insert on each side of the tube being unplugged to hol
123. olyethylene Noryl or cellulose propionate can be autoclaved at 121 C for about 20 minutes Note that autoclaving reduces the lifetime of polycarbonate tubes Also polypropylene 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 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 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 disinfection is expressed or implied When sterilization or disinfection is a concern consult your laboratory safety officer regarding proper methods to use LR IM 24AC Table 7 1 Tube and Bottle Sterilization and Disinfection Care and Maintenance Tube Bottle and Accessory Care ic amp v v v S 2 E gt S 3 D E S e v g 2 3o 3 v2 sg 5 T os a S 9 Sa es 223 592 2
124. omponents that do not band under separation 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 hydrostatic forces generated by centrifugation 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 finished with black polyurethane paint Titanium buckets and lids of high performance rotors are usually painted red for identification LR IM 24AC Rotors 1 Rotor Selection 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
125. on speed changes and deceleration and displays this as a continuously updated value of b o dt B 3 t 1 There are two kinds of experiments in which the integrator is particularly useful duplicating conditions in a series of rate runs and calculating sedimentation coefficients for rate zonal studies To duplicate band positions use the integrator to automatically terminate the run at a preselected value of wt In this way even if the set run speed or acceleration is changed for a rotor band positions will be reproducible For determining sedimentation coefficients the value of w t LR IM 24AC B 1 Use of the w2t Integrator Reproducing Band Positions Refer to Figure B 1 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 swt 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 wt 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 partic
126. on 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 CHAPTER 4 e Polycarbonate and Polypropylene Bottles Speed limitations are required to prevent the bottle material from overstressing and deforming refer to CHAPTER 2 e 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 in CHAPTER 2 Consult individual rotor manuals for allowable run speeds e Stainless Steel Tubes Reduce run speed when centrifuging stainless 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 LR IM 24AC 1 17 1 Rotors General Operating Information 1 18 LR IM 24AC CHAPTER 2 Tubes Bottles and Accessories
127. or components longer than the minimum time specified in the IsoClean usage instructions 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 If the rotor or other components are contaminated with toxic or pathogenic materials follow appropriate decontamination procedures as outlined by appropriate laboratory safety guidelines and or other regulations Consult APPENDIX A to select an agent that will not damage the rotor Sterilization and Disinfection When sterilization or disinfection is a concern consult your laboratory safety officer regarding proper methods to use While Beckman Coulter has tested the following methods and found that In U S contact Biodex Medical Systems Shirley New York internationally contact the U S office to find the dealer closest to you t 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 7 3 7 Care and Maintenance Rotor Care they do not damage the rotor or components no guarantee of sterility or disinfection is expressed or implied Rotors and most rotor components except those made of
128. ormities before using them Do not use a tube that has become yellowed or brittle with age or excess exposure to ultraviolet light Crazing the appearance of fine cracks on tubes and bottles is the result of stress relaxation If a crack approaches the outer wall of the tube or bottle discard it Discard any deformed or cracked adapters 7 9 7 Care and Maintenance Tube Cap Care Tube and Bottle Storage Tubes and bottles have an indefinite shelf life if properly stored Store ina 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 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 approximately 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 If a tube is jammed or collapsed in the rotor try one of the following techniques but DO NOT force the tube Contact your Beckman Coulter Service representative if you
129. ors components and accessories designed for use in the rotor and ultracentrifuge being used 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 rotors may void the rotor warranty and should be prohibited by your laboratory safety officer 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 speeds indicated however speed reductions may be required because of weight considerations of tubes adapters and or the density of the solution being centrifuged Be sure to observe the instructions in the applicable rotor manual NEVER attempt to slow or stop a rotor by hand The strength of containers can vary between lots and will depend on handling and usage We highly recommend that you pretest them 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 containers 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
130. pe 70 Ti 70 000 504 000 44 8 x 38 5 308 G4H R S Type 60 Ti 60 000 362 000 63 8 x 38 5 308 G9 H R S Type 55 2 Ti 55 000 340 000 64 10 x 38 5 385 G9 H R S VC 53 53 000 249 000 36 8 x 39 312 H R S Type 50 2Ti 50 000 302 000 69 12 x 39 468 F G9 H 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 G7 H 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 6x 94 564 H R S LR IM 24AC Table 1 1 Beckman Coulter Preparative Rotors by Use Continued Rotors Rotor Selection Relative Number of Nominal Maximum Centrifugal Tubes x Nominal Rotor For Use in Speed Field xg k Capacity mL Capacity Instruments Rotor rpm at Fmax Factor of Largest Tube mL Classified Type 28 28 000 94 800 393 8x40 320 HERS Rotors for Differential Flotation Type 50 4 Ti 50 000 312 000f 33 44x 6 5 286 G9 H R S Type 50 3 Ti 50 000 223 000 49 18x 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 G4H R S Type 25 25 000 92 5009 62 100 x 1 100 C D F G H R S Rotors for Centrifuging Large Particles Type 70 1 Ti 70 000 450 000 36 12 x 13 5 162 G4H R S Type 50 50 000 196 000 65 10 x 10 100 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 G H Q R S Type 30 30 000 106 000 213 1
131. r Universal Fraction Recovery System 343890 can be useful when recovering sample from tubes see publication L5 TB 081 Capped Tubes 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 LR IM 24AC 3 25 Using Tubes Bottles and Accessories Sample Recovery OptiSeal Tubes Centrifugation exerts high forces on plastic labware The effect of these forces on OptiSeal labware is compression of the tube characterized 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 50 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 1 After centrifugation use the spacer removal tool 338765 or a hemostat to carefully remove the spacers taking care not to scratch the rotor cavities A tube will sometimes come out of the rotor cavity along w
132. r Vertical Tube Rotors Relative Radial Distances Number of Maximum Centrifugal Tube mm Tubes x Tube Rotor Speed Field x g Angle Capacity Type rpm at max degrees max lav nin k Factor mL Vertical Tube VTi 90 90 000 645 000 0 71 1 64 5 57 9 6 8x5 1 VTi 80 80 000 510 000 0 71 1 64 5 57 9 8 8x5 1 VTi 65 2 65 000 416 000 0 87 9 81 3 74 7 10 16x 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 8x5 1 VC 53 53 000 249 000 0 79 0 66 0 53 1 36 8 x 39 VTi 50 50 000 242 000 0 86 6 73 7 60 8 36 8 x 39 VAC 50 50 000 242 000 0 86 6 73 7 60 8 36 8 x 39 Near Vertical Tube NVT 100 100 000 750 000 8 67 0 57 6 48 3 8 8x 5 1 NVT 90 90 000 645 000 8 71 1 61 8 52 4 10 8x5 1 NVT 65 2 65 000 416 000 8 5 87 9 78 4 68 8 15 16x5 1 NVT 65 65 000 402 000 7 5 84 9 72 2 59 5 21 8 x 13 5 a Rotors listed in parentheses are no longer manufactured b Maximum speeds are based on a solution density of 1 7 g mL in all vertical tube and near vertical tube rotors 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
133. r pelleting isopycnic studies separation as a function of density and rate zonal studies separation 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 CHAPTER 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 separations when run time reduction is important Only Quick Seal and OptiSeal tubes are used in vertical tube rotors making tube caps unnecessary Refer to CHAPTER 6 for specific information about the use of vertical tube rotors Near vertical tube rotors are designed for gradient centrifugation when there are components in a sample mixture that do not participate 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
134. rative rotors are provided in the table of general specifications in each rotor use section 1 s dr dt x 1 w2r where dr dt is the sedimentation velocity 1 Rotors Rotor Selection 1 10 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 shortened in some rotors by using partially filled thickwall polypropylene and polycarbonate tubes The k factors for half filled tubes can be calculated by using an approximate rmax and r in k factor equation 1 Figure 1 3 Sedimentation Coefficients in Svedberg Units for Some Common Biological Materials Cytochrome c Collagen Yeast tRNA Albumin Soluble Proteins Luteinizing hormone Immunoglobulin G Aldolase Nucleic Acids Catalase E coli rRNA Calf liver DNA 40 Vesicular stomatitis virus RNA Bacteriophage T5 DNA 60 Bacteriophage T2 amp T4 DNAs Broad bean mottle 20 a Macroglobulin Ribosomal subunits R
135. rin adapters 2 10 LR IM 24AC CHAPTER 3 Using Tubes Bottles and Accessories Introduction This chapter 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 CHAPTER 4 for fixed angle rotors in CHAPTER 5 for swinging bucket rotors and in CHAPTER 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 Added D usually load a tube by allowing the gradient solutions to run down the First side of the tube The heaviest concentration is loaded first followed by successively lighter concentrations This method is acceptable for wettable tubes however loading a nonwettable tube such as 15 Ultra Clear polypropylene and polycarbonate by allowing solutions to run down the side of the tube can cause mixing 10 Added Last 20 Gradients in nonwettable tubes can be prepared using a gradient former by placing a long syringe needle or tubing to the tube bottom and reversing the gradient chambers
136. 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 with 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 Figure C 1 find the curve that corresponds to the desired run temperature 20 and tube fill volume full e The maximum allowable rotor speed is determined from the point where this curve intersects the homogeneous CsCl density 52 000 rpm 2 InFigure C 2 sketch a horizontal line corresponding to each particle s buoyant density 3 Mark the point where each density intersects the curve corresponding to the maximum speed and selected temperature Particles will band at these points along the tube axis C 4 LR IM 24AC The Use of Cesium Chloride Curves Typical Examples for Determining CsCl Run Parameters 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 Wh
137. s aT Delrin Adapters EQ 10 d d d d Ta Tra 7 i 7 sing EQ 11 d d d Paa Dra 5 t L sind 2 cos 0 LR IM 24AC 2 9 2 Tubes Bottles and Accessories Adapters where Tmax the distance in millimeters from the axis of rotation to the farthest part of the tube cavity d outside diameter of the adapter d inside diameter of the adapter L adapter cavity length t thickness of the adapter bottom and o tube angle of the rotor being used The values of r max and 1 min can be used to calculate the k factor and the relative centrifugal field when adapters are used see the equations in the Glossary Table 2 3 Dimensions of Delrin Adapters Delrin Adapter Dimensions mm Tube Size Part d mL Number EP r 0 8 305527 13 08 5 49 42 09 3 99 356860 18 08 5 36 43 51 3 99 2 303376 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 a Use these values to calculate radial distances for tubes in Del
138. s while maximizing separation efficiency HDPE High density polyethylene used for adapters Glossary 1 Glossary Glossary 2 Isopycnic 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 konical tubes Thin walled polypropylene 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 Maximum volume The maximum volume at which a tube should be filled for centrifugation sometimes referred to as maximum fill volume or nominal fill volume Mechanical overspeed cartridge An assembly installed in the bases of some older rotors or swinging bucket rotor adapters as part of the mechanical overspeed protection system Meniscus 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 Near vertical tube rotor A rotor in which the tubes are held at a slight angle usually 7 to 10 degrees Neoprene 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 Noryl Modified thermoplastic polyphenylene
139. s 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 5 8 LR IM 24AC Using Swinging Bucket Rotors Operation Figure 5 2 Checking Hook on Bucket Positions After the Rotor is Installed Removal and Sample Recovery 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 Note the partially unhooked bucket on the right LR IM 24AC 5 9 5 Using Swinging Bucket Rotors Operation 5 10 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 NOTE If conical shaped adapters that support konical tubes are difficult to remove after centrifugation an extractor tool 354468 is available to facilitate removal While pressing the rubber tip against the Extractor adapter wall Tool pull the tool 354468 and adapter up and out of the cavity 5 Refer to CHAPTER 3 for sample recovery m
140. s in centimeters and h is the interband separation in centimeters LR IM 24AC C 5 The Use of Cesium Chloride Curves Typical Examples for Determining CsCl Run Parameters LR IM 24AC APPENDIX D Gradient Materials _ Description This Appendix contains reference information on commonly used gradient materials General instructions for filling and sealing tubes including gradient preparation are contained in CHAPTER 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 e Its density range should be sufficient to permit separation of the particles of interest by the chosen density gradient technique without overstressing the rotor e It should not affect the biological activity of the sample e It should be neither hyperosmotic or hypoosmotic when the sample is composed of sensitive organelles e It should not interfere with the assay technique It should be removable from the purified product It should not absorb in the ultraviolet or visible range e Itshould be inexpensive and readily available more expensive materials should be recoverable for reuse Itshould be sterilizable
141. s 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 precipitating gradient materials and speed reduction for high temperature operation 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 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 if the rotor is used with carriers buckets belts or other devices not of Beckman Coulter manufacture 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 bearings seals and lubrication are excluded from this warranty and should be frequently inspected and replaced if they become worn or damaged Keeping a rotor log is not mandatory but may be desirable for maintenance of good laboratory practices Repair and Replacement Policies 1 If a Beckman Coulter rotor is determined by Beckman Coulter to be defective
142. ses are no longer manufactured b 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 c Relative Centrifugal Field RCF is the ratio of the centrifugal acceleration at a specified radius and speed r 2 to the standard acceleration of gravity g according to the following formula RCF rw 2 g where r is the radius in millimeters is the angular velocity in radians per second 2mRPM 60 and g is the standard acceleration of gravity 9807 mm s2 After substitution RCF 1 12r RPM 1000 2 Class G Model L3 only Except L5 and L5B Maximum RCF measured at outer row Maximum RCF measured at the third outermost row Radial distances are those of the third row zampa 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 Pelleting Differential Separation Pelleting separates particles of different sedimentation coefficients 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 forc
143. sharp tools ona rotor as the surface can 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 LR IM 24AC 7 1 Care and Maintenance Rotor Care Wash rotors and rotor components immediately if salts or other corrosive materials are used or if spillage has occurred Rote Cleaning DO NOT allow corrosive materials to dry on the rotor Kit 339558 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 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 a Useamild 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 e Most laboratory detergents are too harsh for aluminum rotors and components e The Rotor Cleaning Kit 339558 contains two quarts of Solution 555 and brushes that will not scratch rotor surfaces Handle P O Ring T Lid
144. ss fluid with a syringe or pipette e If necessary wipe the inside of the stem with a lintless tissue 3 Fill the remaining tubes in the same manner IN Table 3 2 OptiSeal Tubes and Accessories Size Volume Part Number Rack mm mL pkg 56 Spacer Assembly Rotor 361627 361678 pkg 2 amber Ultem SW 55 Ti aS 13 x 33 3 3 361650 SW 50 1 ss 361621 Bell top 361676 pkg 2 amber Ultem 13x48 4 7 361638 anal 2 5 Type 50 3 Ti 3 5 Using Tubes Bottles and Accessories Filling and Plugging OptiSeal Tubes Table 3 2 OptiSeal Tubes and Accessories Continued Size Volume Part Number Rack mm mL pkg 56 Spacer Assembly Rotor 362198 VTi 90 gold aluminum VTi 80 361638 VTi 65 2 362185 NVT 90 w C NVT 65 2 13x51 4 9 362199 black Noryl 361638 VTi 65 361623 Bell top 361670 pkg 2 Type 90 Ti amber Ultem Type 80 Ti h 16x60 8 9 361642 pe Lm Type 65 Type 50 Ti Type 50 362181 362202 gold aluminum NVT 65 16x70 11 2 gt _ 360538 VTI 65 1 361669 pkg 2 Type 70 Ti amber Ultem Type 60 Ti 361646 Le ae i 361625 Bell top ype 50 2 Ti Type 42 1 Type 30 25x77 32 4 392883 pkg 2 amber Ultem SW 32 Ti 361646 SW 28 362183 362204 gold aluminum VTi 50 25x89 36 2 360542 VAC 50 VC 53 a
145. st the nylon insert Refer to Table 3 3 for detailed information about tube caps Do not interchange tube caps or tube cap components even if they appear to be the same Tube caps are designed specifically for a particular 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 difference 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 Titanium Caps High strength titanium cap assemblies for thinwall Ultra Clear and polypropylene 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 polypropylene 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
146. te the sample symmetrically e Use thickwall tubes partially filled to distribute sample to additional tubes e Layer a low 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 Overspeed Protection LR IM 24AC 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 For example two three four or six tubes can be arranged symmetrically in a six place rotor 1 15 1 Rotors General Operating Information 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 Optima L and LE classified R and Optima XL and L XP classified 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 mo
147. 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 two 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 NOTE Ifa partially filled thickwall tube is centrifuged the tube does not require liquid support and therefore the buffer solution is not required Cesium chloride gradients can be made by filling the tube with a homogeneous solution of CsCl and sample Select a homogeneous CsCl solution density so that when it is distributed its density range will encompass the density of the particle s of interest Refer to APPENDIX C for an explanation of the use of the CsCl curves 3 2 LR IM 24AC Using Tubes Bottles and Accessories General Filling and Sealing or Capping Requirements General Filling and Sealing or Capping Requirements LR IM 24AC See Table 3 1 for general filling and sealing or capping requirements for tubes and bottles used in preparative rotors Maximu
148. trong 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 a Do not lubricate O rings or gaskets Decontamination Coney 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 LR IM 24AC 7 11 Care and Maintenance Tube Cap Care solutions and found two that do not harm anodized aluminum RadCon Surface Spray or IsoClean Solution for soaking and Radiacwash 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 disinfection is expressed or implied Consult your laboratory safety officer regarding the proper decontamination methods to use If tube caps are contaminated with toxic or pathogenic solutions decontaminate or dispose of them as directed by your laboratory safety officer following appropriate safety guidelines Check the chemical resistances list in APPENDIX A to be sure the decontamination method will not damage any part of the rotor
149. tructions 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 CHAPTER 2 for labware selection information and CHAPTER 3 for recommended filling and sealing or capping requirements and for sample recovery procedures Refer to CHAPTER 7 for information on the care of rotors and accessories Description 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 atmospheric pressure inside the rotor during centrifugation if they are properly lubricated Some rotors have fluted bodies designed
150. ube Caps Part Number Torque Value Torque wrench titanium cap 11 mm 7 16 in 10 to 11 Nem 90 to 100 in Ib 858121 341968 335320 titanium Socket 870432 Torque wrench 331151 red 20 mm 3 4 in 11 to 13 6 Nem 100 to 120 in Ib 858121 330901 red 301870 for the first four runs Socket 858122 338905 blue 11 Nem 100 in Ib Socket 858123 338904 black starting with the fifth run 338903 used with Type 21 rotor 20 mm 3 4 in 301870 11 Nem 100 in Ib 3 20 LR IM 24AC Using Tubes Bottles and Accessories Capping Tubes Table 3 4 Required Tools and Torque Values Continued Cap Nut Size Tightening Tool Tube Caps Part Number Torque Value Hex driver 841884 303624 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 a Unless otherwise indicated caps are clear anodized aluminum b Unless otherwise indicated cap nuts are aluminum 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 Nut Delrin Washer O ring lt St d or Gasket AHE Nylon Insert 2 Loosely assemble the stem the O ring or gasket the crown the crown was
151. ugh the filling hole in the stem e Thinwall tubes must be as full as possible to prevent tube collapse 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 The tube must be pushed up past the O ring so that the crown will clamp the tube and NOT the O ring 3 22 LR IM 24AC Using Tubes Bottles and Accessories 3 Filling and Capping Tubes Figure 3 5 Tube Cap Vise 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 f 16 mm 5 s in all other caps Filling and Capping Tubes To prevent spillage and provide support polycarbonate and polypropylene 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 Screw the vise to a bench or table top for operation The vis
152. ular Weight 342 3 D 4 Density Conversion for Cesium and Rubidium Salts at 20 C D 5 Scope _ Scope of this Manual LR IM 24AC 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 CHAPTER 1 describes by usage 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 section is a discussion of rotor materials components and centrifugation techniques CHAPTER 2 describes various tubes bottles adapters and spacers to help you choose a particular tube or bottle for your application CHAPTE

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