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Instructions For Use

1. Some rotors have fluted bodies designed to eliminate unnecessary weight and minimize stresses Tubes Fixed angle rotors can accommodate a variety of tube types listed in the rotor manual Refer to CHAPTER 3 for tube filling and sealing requirements Observe the maximum rotor speeds and fill volumes listed in the applicable rotor manual Rotor Preparation and Loading AN 4 2 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 Make sure that the rotor and lid are clean and show no signs of corrosion or cracking 2 check the chemical compatibilities of all materials used Refer to APPENDIX A J verify that tubes and accessories being used are listed in the applicable rotor manual a TLR IM 9AC Using Rotors Fixed Angle Rotors Rotor Preparation and Loading Be sure that metal threads in the rotor are clean and lightly but evenly lubricated with Spinkote lubricant 306812 a Also ensure that O rings are lightly but evenly coated with silicone vacuum grease 335148 Dry the exterior of the tubes Moisture between the tube and the rotor cavity may lead to tube collapse and increase the force required to extract the tube a Slide the filled and sealed if required tubes into the tube cavities Tubes must be arranged symmetricall
2. Plasma membranes Mitochondria 100 000 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 Rotors Rotor Selection Isopycnic Separations 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 EQ 6 d s Pp Pyg 18u where v sedimentation velocity dr dt d particle diameter py particle density p solution density u viscosity of liquid media g standard acceleration of gravity At equilibrium p p is zero and particle velocity is therefore zero The gradient may be preformed before the run or generated during centrifugation For gradients formed by centrifugation the time it takes to form a gradient depends on the sedimentation and diffusion coefficients of the gradient material the pathlength and the ro
3. Repair and Replacement Policies 1 If a Beckman Coulter rotor is determined by Beckman Coulter to be defective Beckman Coulter will repair or replace it subject to the Warranty Conditions A replacement rotor will be warranted for the time remaining on the original rotor s warranty If a Beckman Coulter centrifuge is damaged due to a failure of a rotor covered by this warranty Beckman Coulter will supply free of charge 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 Warranty 1 Ultracentrifuge Rotor Warranty Warranty 2 Beckman Coulter and ii if the centrifuge is currently covered by a Beckman Coulter warranty or Full Service Agreement all labor necessary for repair of the centrifuge 3 IfaBeckman 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 IfaBeckman 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
4. Vertical Tube and Near Vertical Tube Rotors 1 5 Sedimentation Coefficients in Svedberg Units for Some Com mon Biological Materials 1 11 Arranging Tubes Symmetrically in a Rotor 1 14 Filling OptiSeal Tubes Stems are large enough to accept stan dard pipettes 3 5 The Cordless Quick Seal Tube Topper 3 7 Fixed Angle Rotor 4 2 Swinging Bucket Rotor 4 6 Vertical Tube Rotor 4 12 Near Vertical Tube Rotor 4 12 Precipitation Curves for the MLN 80 Rotor B 4 CsCl Gradients at Equilibrium for the MLN 80 Rotor B 5 Xi Tables Xii Tables 1 1 2 1 3 1 3 2 5 1 5 2 Cl C2 C3 C4 Rotors Used in Beckman Coulter Tabletop Ultracentrifuges 1 6 Characteristics and Chemical Resistances of Tube Materials 2 2 Filling and Capping Requirements for Tubes 3 3 OptiSeal Tubes and Accessories 3 4 Tube Sterilization and Disinfection 5 7 Troubleshooting Chart 5 9 Commonly Used Gradient Materials with Their Solvents C 2 Density Refractive Index and Concentration Data Cesium Chloride at 25 C Molecular Weight 168 37 C 3 Density Refractive Index and Concentration Data Sucrose at 20 C Molecular Weight 342 3 C 4 Density Conversion for Cesium and Rubidium Salts at 20 C C 5 Scope _ Scope of this Manual This manual contains general information for properly preparing a rotor for centrifugation in a Beckman Coulter tabletop preparative ultracentrifuge This manual should be us
5. above and the replacement policy 5 Damage to a Beckman Coulter rotor or instrument due to the failure or malfunction of a non Beckman Coulter tube bottle tube cap spacer or adapter is not covered under this warranty although Beckman Coulter will assist in seeking compensation under the manufacturer s warranty Disclaimer ITIS 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 TH
6. while allowing components that do not band under separation conditions to either pellet to the bottom or float to the top of the tube Like the vertical tube rotors near vertical tube rotors use only Quick Seal and OptiSeal tubes Table 1 1 lists Beckman Coulter tabletop preparative rotors TLR IM 9AC Rotors 1 Rotor Selection Figure 1 3 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 l min max Pathlength Near Vertical Tube Rotors min Tmax Pathlength Dark gray represents pelleted material light gray is floating components and bands are indicated by black lines TLR IM 9AC 1 5 Rotors Rotor Selection Table 1 1 Rotors Used in Beckman Coulter Tabletop Ultracentrifuges Radial Distances Number of mm Tubes x Max Speed Nominal Rotor RCF Capacity Manual Rotor Profile and Description k factor Tmax lav min largest tube Number E MLA 1505 150 000 39 9 27 8 15 8 8x2 0mL 393554 Fixed Angle 1 003 000 x g 30 Angle 10 4 MLA 130 130 000 59 9 41 9 29 9 10x2 0mL TL TB 021 Fixed Angle 1 0
7. 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 431227 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 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
8. 1 Tube Sterilization and Disinfection a se MOGAO a 3 P 3 k 7 s js E D f t x x teh T 2 2 Io gt a 2 o f D I S 5 o Uo ENS o E z ED 5 i a S E D c 9 E EI is S Bs eg gt E g o gt cof cs A S 3 5 gt I 5 SRI se ge SR q 5 iu Ma i aT m o a Ultra Clear no no yes yesd yes yes yes yes no polycarbonate yes no yes yesd no yesf yes yes no polypropylene yes no yes yes yes yes9 yesh yes no polyethylene no no yes yes yes yes yes yes yes cellulose no no no no no yes yes yes no propionate This information is provided as a guide to the use of sterilization and disinfection techniques for tube materials Cold sterilization results shown are for short duration 10 minute soak periods reactions may differ with extended contact Refer to Appendix A of this manual for information about specific solutions To avoid deformation autoclave tubes open end down in a tube rack at 15 psig for no more than 20 minutes allow to cool before removing from tube rack DO NOT autoclave capped or sealed tubes Flammable do not use in or near operating ultracentrifuges 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 Below 26 C only Below 21 C only While Beckman Coulter has tested these methods and found that they do
9. 4 12 TLR IM 9AC Using Rotors 4 Vertical Tube and Near Vertical Tube Rotors 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 plugs gaskets and spacers are clean and show no signs of corrosion or cracking The high forces generated in these rotors can cause damaged components to fail 2 Check the chemical compatibilities of all materials used Refer to APPENDIX A J verify that tubes and accessories being used are listed in the applicable rotor manual Rotor Preparation and Loading Be sure that plug threads are clean and lightly but evenly lubricated with Spinkote lubricant 306812 Gasket Threads b Oo 2 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 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 Tubes must be arranged symmetrically in the rotor see Figure 1 5 TLR IM 9AC 4 13 Using Rotors Vertical Tube and Near Vertical Tube Rotors Opposing tubes must be filled to the same level with liquid of the same density Refer to Rotor Balance
10. 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 TLR IM 9AC C 5 C Gradient Materials Introduction C 6 TLR IM 9AC List of References APPENDIX D References Documents referenced below can be obtained by calling Beckman Coulter at 1 800 742 2345 in the United States or by contacting your local Beckman Coulter office IN 175 IN 181 IN 189 IN 192 IN 197 L5 TB 081 TL TB 008 Chemical Resistances for Beckman Coulter Centrifugation Products How to Use Quick Seal Tubes with the Beckman Cordless Tube Topper Using OptiSeal Tubes Use and Care of Centrifuge Tubes and Bottles Rotor Safety Multi lingual Beckman Fraction Recovery Systems Instructions for Using the Beckman Coulter CentriTube Slicer Documents referenced below are available upon request from Beckman Coulter Inc Marketing Communications 250 S Kraemer Blvd Brea CA 92821 U S A or are available at www beckmancoulter com A 1790 A 1804 A 1846 A 1850 A 1851 BA99 60495 BR 10656 Plasmid Separations in NVT Near Vertical Tube Rotors Rapid Mitochondrial DNA Isolation Using Beckman Type 100 Ti Rotor Selected Run Conditions for Optimizing the Separation of RNA Using Centrifugation in Either a Preparative Floor or Tabletop Instrument A Rapid Method for Ribosome Preparation Part 1 Using High Capa
11. Glossa ry 4 TLR IM 9AC TLR IM 9AC Ultracentrifuge Rotor Warranty All Beckman Coulter ultracentrifuge Fixed Angle Vertical Tube Near Vertical Tube Swinging Bucket and Airfuge rotors are warranted against defects in materials or workmanship for the time periods indicated below subject to the Warranty Conditions stated below Preparative Ultracentrifuge Rotors 5 years No Proration Analytical Ultracentrifuge Rotors 5 years No Proration ML and TL Series Ultracentrifuge 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 c
12. Preparation DNA from Bacteriophage Lambda Isolated in the TL 100 Tabletop Ultracentrifuge TLA 100 4 Rotor New Rotor Increases Sample Volume Plasmid Separations in Tabletop Ultracentrifuges Using Fixed Angle Rotors Determination of Molecular Weights by Sedimentation Equilibrium Short Column Sedimentation Equilibrium Analysis for Rapid Characterization of Macromolecules in Solution Isolation of Human VLDL LDL HDL and Two HDL Subclasses in the TL 100 Tabletop Centrifuge Using the TLA 100 4 Rotor Plasmid Separations in Just 1 1 2 Hours With the New TLN 120 Rotor MLA 55 Rotor for Optima MAX Series Micro Ultracentrifuges Using k Factor to Compare Rotor Efficiency Ultracentrifuge Rotors Brochure Rapid Isolation of Both RNA amp DNA from Cultured Cell Purity Antigenicity and Immunogenicity Separation of Lipoproteins from Human Plasma with the TLN 100 Near Vertical Tube Rotor Separation of Plasma Lipoproteins with the TLN 100 Near Vertical Tube Rotor TLR IM 9AC Angular velocity Rate of rotation measured in radians per second _ 2nrpm 0 700 or 0 10472 rpm 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 in a specified liquid medium Buna N Black nitrile rubber used for O rings a
13. 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 75 TLR IM 9AC 80 The Use of Cesium Chloride Curves B Typical Examples for Determining CsCl Run Parameters Figure B 2 CsCI Gradients at Equilibrium for the MLN 80 Rotor 1 90 1 80 Z 4o 900 Pi 09 A vore goo 50 S 1 70 RS QS 40 000 e E eS S AA STA hy 9 999 i zd 1 60 zy E D gt a 1 50 M3 S 4 S of d 140 qe MLN 80 ROTOR 4 30 20 C 4C 1 20 37 0 42 0 47 0 52 0 Distance from Axis of Rotation mm Centrifugation of homogeneous CsCl solutions at the maximum allowable speeds from Figure B 1 results in gradients presented here TLR IM 9AC B 5 The Use of Cesium Chloride Curves Typical Examples for Determining CsCl Run Parameters TLR IM 9AC APPENDIX C Gradient Materials _ 00 Introduction This Appendix contains reference information on commonly used gradient materials General in
14. Top Tubes TLR IM 9AC 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 Ultra Clear For swinging bucket rotors fill to within 2 or 3 mm of the top of the tube Fill all opposing tubes to the same level Polypropylene Fill all opposing tubes to the same level For swinging bucket rotors fill to within 2 or 3 mm of the top of the tube Fillthickwall polypropylene tubes at least half full to maximum level in fixed angle rotors Speed reduction is required Refer to the applicable rotor manual 3 11 3 Using Tubes and Accessories Sample Recovery Polyethylene For swinging bucket and fixed angle rotors fill these tubes from half full to maximum level Refer to the applicable rotor manual 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 required The Beckman Coulter Fraction Recovery System 342025 and adapter 347828 can be us
15. amber Ultem TLA 110 mm m a Spacers and plugs are shown in the correct orientation for placement onto tubes b Disposable plastic plugs included C Ultem is a registered trademark of GE Plastics 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 to wet the tube a Homogeneous solutions of gradients and sample may be loaded into the tubes and centrifuged immediately See Gradient Preparation above b Ifthe 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 3 4 TLR IM 9AC Using Tubes and Accessories 3 Filling and Plugging OptiSeal Tubes 2 After filling the tube make sure that there is no fluid in the stem a Draw off excess fluid with a syringe or pipette b If necessary wipe the inside of the stem with a lintless tissue 3 Fill the remaining tubes in the same manner b Figure 3 1 Filling OptiSeal Tubes Stems are large enough to accept standard pipettes Stem Base Sealing the Tube Plugs Ei
16. 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 Beckman Coulter Field Service if you 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 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 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 Returning a Rotor or Accessory to the Factory 5 8 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 r
17. 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 Temperature Limits Each labware material has a specified temperature range Although some ultracentrifuges can achieve temperatures as high as 45 C only certain tube or bottle materials can be run under these conditions Most containers are made of thermoplastic materials that soften at elevated temperatures This temperature induced softening together with such factors as the centrifugal force the run duration the type of rotor previous run history and the tube angle can cause labware to collapse Therefore if high temperature runs above 25 C are required it is best to pretest labware under the actual experimental conditions using buffer or gradient of similar density rather than a valuable sample 25 C Qmm J 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 Spacer and Floating Spacers 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 one o
18. in CHAPTER 1 1 Place filled tubes in at least two opposing cavities 4 itis important that each cavity being used is completely filled Usethe required spacers and or floating spacers if necessary to complete the loading operation a IfOptiSeal tubes are being used install a spacer over each plugged tube refer to the applicable rotor manual ue Spacer C Tube Plug _ Tube 1 Leave cavities without tubes completely empty 4 14 TLR IM 9AC Using Rotors Vertical Tube and Near Vertical Tube Rotors b IfQuick Seal tubes are being used install spacer and or floating spacers over sealed tubes refer to the applicable rotor manual Spacers E i Floating Spacer a Banai Bell Top Tube Tube 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 D Inserta rotor plug with the white gasket end down over each spacer screw in the plug 6 Using the plug adapter and torque wrench listed in the rotor manual torque each rotor plug to 13 6 Nem 120 in Ib a Toavoidstripping the plugs apply downward pressure to the adapter while tightening the plugs Donotovertighten plugs Remove the rotor fr
19. 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 Place the tubes back into the tube rack Openings in the rack allow the tubes to be pierced either from the bottom or sides permitting fractions to be easily collected regardless of the type of separation NOTE If you plan to collect particles from the tube side or bottom first create an air passage by removing the tube plug see instructions below or inserting a hollow hypodermic needle in the top of the tube TLR IM 9AC 3 13 3 Using Tubes and Accessories Sample Recovery 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 Take care when p
20. other than maximum rated speed by use of the following formula EQ4 2 rated speed of rotor Kadj actual run speed 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 EQ 5 ec oec F s k ky where the k factors have been adjusted for the actual run speed used Figure 1 4 lists sedimentation coefficients for some common biological materials The k factors at maximum speeds for Beckman Coulter preparative rotors are provided in Table 1 1 1 10 TLR IM 9AC TLR IM 9AC Rotors Rotor Selection Figure 1 4 Sedimentation Coefficients in Svedberg Units for Some Common Biological Materials Cytochrome c Collagen Yeast tRNA Albumin Luteinizing hormone Soluble Proteins Immunoglobulin G Aldolase Nucleic Acids Catalase a Macroglobulin E coli rRNA 20 calf liver DNA Ribosomal subunits 40 Vesicular stomatitis virus RNA Bacteriophage T5 DNA 60 Bacteriophage T2 amp T4 DNAs Broad bean mottle Ribosomes Poliomyelitis 200 Tobacco mosaic Equine encephalitis Polysomes Viruses Rous sarcoma 800 Feline leukemia Bacteriophage T2 Microsomes Subcellular Particles
21. 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 When the seal former cools remove it by hand or with the removal tool 361668 1 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 2 The tube is now ready for centrifugation Seal the remaining tubes 3 Return the Tube Topper to its charging stand when finished 3 10 TLR IM 9AC Using Tubes 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 e Thinwall Tubes Fill to within 2 or 3 mm of the top for 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 or 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
22. 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 765 and 250 F Noryl Modified thermoplastic polyphenylene 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 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 ba
23. 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 Using Tubes and Accessories Making Ultra Clear Tubes Wettable 3 18 TLR IM 9AC CHAPTER 4 Using Rotors M Introduction This section contains instructions for using rotors in tabletop 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 5 for information on the care of rotors and accessories 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 b
24. to float and collapse Ensure that tubes and tube cavities or buckets are dry before inserting the tubes Reagent used that attacks the tube material Refer to APPENDIX A for chemical compatibilities of tube material and chemicals Tubes run above their rated speed Refer to the applicable rotor manual for maximum speeds TLR IM 9AC APPENDIX A Chemical Resistances for Beckman Coulter Centrifugation Products List of Chemical Resistances Appendix A is replicated in the separate pdf document Chemical Resistances IN 175 TLR IM 9AC A 1 Chemical Resistances for Beckman Coulter Centrifugation Products List of Chemical Resistances A 2 TLR IM 9AC APPENDIX B The Use of Cesium Chloride Curves _ Introduction TLR IM 9AC This Appendix describes how to determine a maximum rotor speed and the final band positions of particles when performing isopycnic separations using cesium chloride gradients The examples shown here are for the MLN 80 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
25. 19 000 x g 28 Angle 8 7 m TLN 1209 120 000 363 30 3 24 3 8x1 2mL TL TB 017 Near Vertical 585 000 x g Tube 7 8 Angle p e TLA 120 28 120 000 38 9 318 245 10x2 0mL TL TB 016 Fixed Angle 627 000 xg a 30 Angle 16 TLA 120 1 120 000 38 9 31 8 24 5 14x0 5 mL TL TB 015 Fixed Angle 627 000 x g a 30 Angle 8 TLA 1108 110 000 485 37 2 26 0 8x5 1 mL TL TB 019 J Fixed Angle 657 000 x g 28 Angle 20 TLN 100 100 000 40 2 31 6 23 1 8 x 3 9 mL TL TB 013 Near Vertical 450 000 x g Tube 14 9 Angle TLA 100 4f 100 000 48 5 37 2 26 0 8x5 1mL TL TB 014 Fixed Angle 543 000 x g 28 Angle 16 TLR IM 9AC TLR IM 9AC Rotors Rotor Selection Table 1 1 Rotors Used in Beckman Coulter Tabletop Ultracentrifuges Continued Radial Distances Number of mm Tubes x Max Speed Nominal Rotor RCF Capacity Manual Rotor Profile and Description k factor Tmax av min largest tube Number TLA 100 3 100 000 48 3 37 9 27 5 6x3 5mL TL TB 011 Fixed Angle 541 000 x g 30 Angle 14 E TLA 100 2 100 000 38 9 31 8 245 10x2 0mL TL TB 005 Fixed Angle 436 000 x g 30 Angle 12 TLA 100 1f 100 000 38 9 31 8 24 5 12x0 5mL TL TB 004 Fixed Angle 436 000 x
26. 64 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 23 271 3 1 647 1 699 1 3992 56 951 4 5 651 1 2164 1 3539 24 291 9 1 734 1 720 1 4012 57 980 4 5 823 1 2275 1 3550 25 306 9 1 823 1 741 1 4032 58 1009 8 5 998 1 2387 1 3561 26 322 1 1 913 1 763 1 4052 59 1040 2 6 178 1 2502 1 3572 27 337 6 2 005 1 7846 1 4072 60 1070 8 6 360 1 2619 1 3584 28 353 3 2 098 1 808 1 4093 61 1102 9 6 550 1 2738 1 3596 29 369 4 2 194 1 831 1 4115 62 1135 8 6 746 1 2858 1 3607 30 385 7 2 291 1 856 1 4137 63 1167 3 6 945 1 298 1 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
27. 99 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 and refractive index data are from the International Critical Tables b Divide by 10 0 to obtain w v C 4 TLR IM 9AC Gradient Materials Introduction Table C 4 Density Conversion for Cesium and Rubidium Salts at 20 C w w CsCl CsBr CsI Cs S04 CsNO RbCl RbBr Rbi Rb2504 RbNO3 1 1 00593 1 00612 1 00608 1 0061 1 00566 1 00561
28. Allowable Run Speeds below The ultracentrifuge identifies rotor speed during the run by means of a magnetic speed sensor system in the rotor chamber of the instrument and magnets on the bottom of the rotor The overspeed system ensures that the rotor does not exceed its permitted speed Forexample two three four or six tubes can be arranged symmetrically in a six place rotor TLR IM 9AC 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 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 B 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 ofthe tube contents to be centrifuged Cesium Chlor
29. D25 12 6483 for densities between 1 00 and 1 37 and p2 gt 10 8601 1D25 13 4974 for densities above 1 37 Bruner and Vinograd 1965 C Divide by 10 0 to obtain 96 w v TLR IM 9AC C 3 C Gradient Materials Introduction Table C 3 Density Refractive Index and Concentration Data Sucrose at 20 C Molecular Weight 342 34 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 0 9982 1 3330 0 1 1463 1 3883 34 389 7 1 138 1 0021 1 3344 1 10 0 0 029 1 1513 1 3902 35 403 0 1 177 1 0060 1 3359 2 20 1 0 059 1 1562 1 3920 36 416 2 1 216 1 0099 1 3374 3 30 3 0 089 1 1612 1 3939 37 429 6 1 255 1 0139 1 3388 4 40 6 0 119 1 1663 1 3958 38 443 2 1 295 1 0179 1 3403 5 50 9 0 149 1 1713 1 3978 39 456 8 1 334 1 0219 1 3418 6 61 3 0 179 1 1764 1 3997 40 470 6 1 375 1 0259 1 3433 7 71 8 0 210 1 1816 1 4016 41 484 5 1 415 1 0299 1 3448 8 82 4 0 211 1 1868 1 4036 42 498 5 1 456 1 0340 1 3464 9 93 1 0 272 1 1920 1 4056 43 512 6 1 498 1 0381 1 3479 10 103 8 0 303 1 1972 1 4076 44 526 8 1 539 1 0423 1 3494 11 114 7 0 335 1 2025 1 4096 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 5
30. E ROTOR AND ACCESSORIES ARE NON RADIOACTIVE NON PATHOGENIC NON TOXIC AND OTHERWISE SAFE TO SHIP AND HANDLE IS REQUIRED TLR IM 9AC www beckmancoulter com Osoon COULTER 2014 Beckman Coulter Inc All Rights Reserved
31. Instructions For Use Rotors and Tubes For Beckman Coulter Tabletop Preparative Ultracentrifuges TLR IM 9AC February 2014 Beckman Coulter Inc 250 S Kraemer Blvd C ull Brea CA 92821 USA OULTER Rotors and Tubes For Beckman Coulter Tabletop Preparative Ultracentrifuges TLR IM 9AC 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 DANGE
32. R 4 viii Temperature Limits 2 6 Spacer and Floating Spacers 2 6 Adapters 2 7 Using Tubes and Accessories 3 1 Introduction 3 1 Gradient Preparation 3 1 Cesium Chloride Gradients 3 2 General Filling and Sealing or Capping Requirements 3 2 Filling and Plugging OptiSeal Tubes 3 4 Filling the Tubes 3 4 Sealing the Tube Plugs 3 5 Filling and Sealing Quick Seal Tubes 3 6 Method A With the Seal Guide 3 9 Method B Without the Seal Guide 3 10 Filling Open Top Tubes 3 11 Open Top Polypropylene Tubes 3 11 Swinging Bucket Rotors 3 11 Fixed Angle Rotors 3 11 Other Open Top Tubes 3 11 Polycarbonate 3 11 Ultra Clear 3 11 Polypropylene 3 11 Polyethylene 3 12 Sample Recovery 3 12 Open Top Tubes 3 12 OptiSeal Tubes 3 12 Removing Plugs from Tables 3 15 Quick Seal Tubes 3 16 Making Ultra Clear Tubes Wettable 3 17 Using Rotors 4 1 Introduction 4 1 Fixed Angle Rotors 4 1 Description 4 1 Tubes 4 2 Rotor Preparation and Loading 4 2 Prerun Safety Checks 4 2 Rotor Preparation and Loading 4 3 Operation 4 4 Installing the Rotor 4 4 Removal and Sample Recovery 4 5 Swinging Bucket Rotors 4 6 CHAPTER 5 APPENDIX A APPENDIX B Contents Description 4 6 Tubes 4 7 Rotor Preparation and Loading 4 7 Prerun Safety Checks 4 7 Rotor Preparation and Loading 4 7 Operation 4 9 Installing the Rotor 4 9 Removal and Sample Recovery 4 10 Vertical Tube and Near Vertical Tu
33. R indicates an imminently hazardous situation 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 TLR IM 9AC iii Safety Notice Chemical and Biological Safety Chemical and Biological Safety Normal ope
34. TE lsoClean can cause fading of colored anodized surfaces Use it only when necessary and do not soak rotor components longer than the minimum time specified in the IsoClean usage 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 Ifthe rotor or other components are contaminated with toxic or pathogenic materials follow EC appropriate decontamination procedures as outlined by appropriate laboratory safety QZ 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 they do not damage the rotor or components no guarantee of sterility or disinfection is expressed or implied gloss Rotors and most rotor components except those made of Noryl can be autoclaved at 121 C for B up to an hour Remove the lid bucket caps or rotor plugs and place the rotor and or buckets i in the autoclave upside down O rings and gaskets can be left in place on the rotor B Ethanol 70 may be used on all rotor components including those mad
35. afely centrifuged at that speed and temperature increases as the fill volume decreases The curves in Figure B 2 show gradient profiles at equilibrium Each curve was generated for the specific rotor speed shown using the maximum CsCl density from Figure B 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 B 2 can be used to approximate B 1 The Use of Cesium Chloride Curves Typical Examples for Determining CsCl Run Parameters 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 NOTE The curves in Figures B 1 and B 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 Typical Examples for Determining CsCl Run Parameters B 2 Example A Knowing homogeneous CsCl solution density 1 63 g mL and approximate particle buoyant densities 1 70 and 1 65 g mL where will particles band 4 b In Figure B 1 find the curve that corresponds to the required run temperature 20 C and fill volume full The maximum allowable rotor speed is determined from the point where this curve inter
36. alternate the placement positions 1 and 3 then 2 and 4 to ensure even wear on the rotor Remember all four buckets must be attached to the rotor whether they are loaded or empty Attach the buckets to the rotor before installing it in the instrument Trying to attach them after the rotor is installed may cause damage to the drive shaft Operation For runs at other than room temperature refrigerate or warm the rotor beforehand for fast equilibration Installing the Rotor To install the rotor carefully lift it with both hands and place it on the drive hub n ss pl yy Ww Ny QF 2 TLseries rotors lock the rotor in place by gently pressing the plunger down until you feel it click When you remove your finger the plunger will remain flush with the rotor body if it is properly engaged TLR IM 9AC 4 9 Using Rotors Swinging Bucket Rotors a Ifthe plunger pops up repeat the procedure Plunger Locked In all tabletop ultracentrifuge models except the Optima MAX XP MAX TL MAX and MAX E it is very important to lock the rotor in place before beginning the run to ensure that the rotor remains seated during centrifugation Failure to lock the rotor in place before beginning the run may result in damage to both rotor and instrument J Refer to the instrument instruction manual for ultracentrifuge operation b Removal and Sample Recovery If disassembly reveals evidence of leakage you shoul
37. and plugged full and heat sealed Ultra Clear open top tubes Quick Seal tubes Polycarbonate thickwall tubes Cellulose Propionate within 2 3 mm of top at least 1 2 full full and capped full and heat sealed 1 2 full to max capless level full and heat sealed tubes full 1 2 full to max capless level no cap Polypropylene at least 1 2 full tubes 1 2 full to max capless level Polyethylene tubes at least 1 2 full 1 2 full to max capless level 3 3 3 Using Tubes and Accessories Filling and Plugging OptiSeal Tubes Filling and Plugging OptiSeal Tubes OptiSeal tubes are not sealed prior to centrifugation a Noryl plug furnished with each tube is inserted into the stem of filled tubes When the tubes are loaded into the rotor with tube spacers and rotor plugs in vertical tube and near vertical tube rotors in place the g force during centrifugation ensures a tight reliable seal that protects your samples For a detailed discussion on the use of OptiSeal tubes refer to Using OptiSeal Tubes publication IN 189 Filling the Tubes For filling convenience use the appropriate eight tube rack listed in Table 3 2 Table 3 2 OptiSeal Tubes and Accessories Size Volume Part Number Rack mm mL pkg 56 Spacer Assembly Rotor 13x 33 3 3 361627 361698 pkg 2 361650 TLN 100 gold aluminum BEN 13 x 48 4 7 361621 Bell top 361676 pkg 2 361638 TLA 100 4
38. are 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 describes the use of cesium chloride curves APPENDIX C contains reference information on some commonly used gradient materials APPENDIX D lists references for further reading Glossary provides a glossary of terms TLR IM 9AC xiii Scope Scope of this Manual xiv TLR IM 9AC CHAPTER 1 Rotors _ Introduction This section is an introduction to the Beckman Coulter family of tabletop 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 Care and maintenance information for all of these rotors is contained in CHAPTER 5 General Description Rotor Designations TLR IM 9AC Beckman Coulter tabletop preparative rotors are named according to the type of rotor and the rotor s maximum allowable revolutions per minute in thousands referred to as rated speed For example the TLS 55 is a swinging bucket rotor with a maximum speed of 55 000 rpm Decimal units that are sometimes part of the rotor name as in the TLA 120 2 and
39. ased on a number of factors Thecentrifugation technique to be used including the rotor in use volume of sample to be centrifuged need for sterilization importance of band visibility and so forth Chemical resistance the nature of the sample and any solvent or gradient media Temperature and speed considerations Whether tubes are to be reused Table 2 1 contains an overview of some of the characteristics of tube materials NOTE This information has been consolidated from a number of sources and is provided only as a guide to the selection of tube or bottle materials Soak tests at 1 g at 20 C established the data for most of the materials reactions may vary underthe 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 Acomplete list of tubes 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 TLR IM 9AC 2 1 Tubes and Accessories Labware Selection Criteria Table 2 1 Characteristics and Chemical Resistances of Tube Materials 2 4 o D o E amp 5 z 5 g g g amp S U 2 S CHIRC 4 ED S 5 D A u u E E o o e 2 S S i o o 3 5 S amp v 2 2 lt Fe e leeds aae ale e le 5 S Me aise ae sc iren ce alan
40. ature refrigerate or warm the rotor beforehand for fast equilibration Installing the Rotor Use an absorbent towel to wipe off condensation from the rotor then carefully place the rotor on the drive hub 2 TLseries rotors Lock the rotor in place by gently pressing the plunger down until you feel it click a When you remove your finger the plunger will remain flush with the rotor body if it is properly engaged 4 4 TLR IM 9AC TLR IM 9AC Using Rotors Fixed Angle Rotors b Ifthe plunger pops up repeat the procedure Plunger Engaged The Optima MAP XP MAX TL MAX and MAX E ultracentrifuges automatically secure the rotor to the drive shaft without the need for engaging the plunger In all tabletop ultracentrifuge models except the Optima MAX XP MAX TL MAX and MAX E it is very important to lock the rotor in place before beginning the run to ensure that the rotor remains seated during centrifugation Failure to lock the rotor in place before beginning the run may result in damage to both rotor and instrument J Refer to the instrument instruction manual for ultracentrifuge operation N 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 TL series rotors To release the plunger at the end of the run gently press it down until you feel it cli
41. be Rotors 4 11 Vertical Tube Rotors Description 4 11 Near Vertical Tube Rotors Description 4 12 Tubes 4 12 Rotor Preparation and Loading 4 13 Prerun Safety Checks 4 13 Rotor Preparation and Loading 4 13 Operation 4 16 Installing the Rotor 4 16 Removal and Sample Recovery 4 17 Care and Maintenance 5 1 Introduction 5 1 Rotor Care 5 1 Cleaning 5 1 Decontamination 5 3 Sterilization and Disinfection 5 3 Inspection 5 3 Field Rotor Inspection Program 5 5 Lubrication 5 5 Tube and Accessory Care 5 5 Cleaning 5 5 Decontamination 5 6 Sterilization and Disinfection 5 6 Inspection 5 7 Tube and Bottle Storage 5 8 Removing Jammed or Collapsed Tubes 5 8 Returning a Rotor or Accessory to the Factory 5 8 Diagnostic Hints 5 9 Chemical Resistances for Beckman Coulter Centrifugation Products A 1 List of Chemical Resistances A 1 The Use of Cesium Chloride Curves B 1 Introduction B 1 Typical Examples for Determining CsCl Run Parameters B 2 Contents APPENDIX C APPENDIX D Gradient Materials C 1 Introduction C 1 References D 1 List of References D 1 Glossary Ultracentrifuge Rotor Warranty Illustrations Illustrations 1 1 1 2 13 1 4 L5 3 1 did 4 1 4 2 4 3 4 4 B 1 B2 Fixed Angle Swinging Bucket Vertical Tube and Near Verti cal Tube Rotors 1 2 Plunger Mechanism in Locked and Released Positions 1 3 Particle Separation in Fixed Angle Swinging Bucket
42. city Fixed Angle MLA 80 Rotor in an Optima MAX Tabletop Ultracentrifuge A Rapid Method for Ribosome Subunit Isolation Part 2 Using the High Capacity Swinging Bucket MLS 50 Rotor in an Optima MAX Tabletop Ultracentrifuge Rotor Safety Guide Warranty and Care Optima MAX XP Benchtop Ultracentrifuge For detailed information on a rotor see the applicable individual rotor manual TLR IM 9AC D 1 References List of References BR 8101 BR 8108 BR 8240 DS 468 DS 514 DS 528 DS 640 DS 641 DS 642 DS 644 DS 670 DS 694 DS 709 DS 728 DS 733 DS 803 DS 814 DS 820 DS 835 DS 850 DS 888 DS 10091 DS 9271 SB 778 SR 171 SR 182 T 1734 T 1784 D 2 Ultracentrifuge Rotors Tubes amp Accessories Catalog Optima Series Optima MAX System Techniques of Preparative Zonal and Continuous Flow Ultracentrifugation Ultracentrifuge Methods for Lipoprotein Research Use of the wt Integrator Formation of Linear Sucrose Gradients for the TLS 55 Rotor A Microscale Method for Isolating Plasmid DNA in the TL 100 Tabletop Ultracentrifuge A Rapid Method for Isolating Plasmid DNA in the TL 100 Tabletop Ultracentrifuge Rate Zonal Separation on Sucrose Density Gradients in the TLS 55 Rotor Rapid Preparation of Synaptic Membranes From Ultrasmall Samples 30 Minute 2 Step Purification of Plasma Membranes from Cultured Cells g Max System Short Pathlengths in High Force Fields Optimizing Centrifugal Separations Sample Loading
43. ck When you remove your finger the plunger will pop up to its released position Plunger Released 2 Remove the rotor from the ultracentrifuge and place it in the rotor vise 4 5 4 Using Rotors Swinging Bucket Rotors J Removethe lid by unscrewing it to the left counterclockwise 4 Use a tube removal tool to remove the spacers and tubes a Swinging Bucket Rotors Description Swinging bucket rotors see Figure 4 2 are most frequently used for density gradient separations either isopycnic or rate zonal Refer to Table 1 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 gt and rotor body positions are numbered for operator convenience Each bucket is sealed by a lubricated O ring between the bucket and the bucket cap When not in the instrument the rotor body must be supported on its rotor stand to permit the buckets to hang properly Figure 4 2 Swinging Bucket Rotor MLA 130 Axis of Rotation 4 6 TLR IM 9AC Using Rotors 4 Swinging Bucket Rotors Tubes 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 requ
44. d assume that some fluid escaped the rotor Apply appropriate decontamination procedures to the centrifuge and accessories TLseries rotors to release the plunger at the end of the run gently press it down until you feel it click When you remove your finger the plunger will pop up to its released position Plunger Unlocked 2 Remove the rotor from the ultracentrifuge and return it to its stand J Detach the buckets from the rotor body 4 10 TLR IM 9AC Using Rotors 4 Vertical Tube and Near Vertical Tube Rotors 4 unscrew the bucket caps then use the appropriate removal tool to remove the tubes 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 N adapter wall Extractor pull the tool Tool and adapter 354468 up and out of the cavity D Refer to CHAPTER 3 for sample recovery methods b Vertical Tube and Near Vertical Tube Rotors 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 1 1 for general rotor specifications Vertical Tube Rotors Description Tubes in vertical tube rotors see Figure 4 3 are held parallel to the axis of rotation in numbered tube cavities T
45. e Polycarbonate tubes are used 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 fluid to escape These tubes have good tolerance to all gradient media except alkalines pH greater than 8 They are satisfactory for some weak acids but are unsatisfactory for all bases alcohol and other organic solvents Polypropylene Tubes 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 go
46. e by allowing solutions to 15 run down the side of the tube can cause mixing 10 mw Gradients in nonwettable tubes can be prepared using a gradient former by placing Last 20 a long syringe needle or tubing to the tube bottom and reversing the gradient chambers In that way the lightest gradient concentration is loaded first underlayed by increasingly heavier concentrations Acomplete 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 TLR IM 9AC 3 1 Using Tubes and Accessories General Filling and Sealing or Capping Requirements 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 form a linear gradient is to allow a step gradient to diffuse to linearity Depending on the concentration differential between steps and the cross sectional area allow 3 to 6 hours for diffusion at room temperature and about 16 hours at 0 to 4 C For diffusion of step gradient in Quick Seal and capped straightwall tubes slowly lay the tube on its
47. e 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 Asample 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 3 6 TLR IM 9AC Using Tubes 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 held Tube Topper Use the applicable tube rack listed in the applicable rotor manual 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 Figure 3 2 The Cordless Quick Seal Tube Topper Charging Stand Remove the Tube Topper from the charging stand a Leave the pushbutton 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 t
48. e 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 Fixed Angle Rotors Description Fixed angle rotors see Figure 4 1 are general purpose rotors that are especially useful for pelleting and isopycnic separations Refer to Table 1 1 for general rotor specifications Tubes in fixed angle rotors 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 compared to swinging bucket rotors resulting in reduced run times Fixed angle rotors have lids with O rings made of Buna N rubber The O rings help to maintain atmospheric pressure inside the rotor during centrifugation if they are properly lubricated TLR IM 9AC 4 1 Using Rotors Fixed Angle Rotors Figure 4 1 Fixed Angle Rotor I min Tav Tmax MLA 130 Axis of Rotation Each fixed angle rotor is specially designed with a fluid containment annulus located below the O ring sealing surface The annulus retains fluid that may escape from leaking or overfilled tubes thereby preventing the liquid from escaping into the instrument chamber Fluid Containment Annulus D N Rotor Body
49. e decontamination procedures to the centrifuge and accessories TLseries rotors to release the plunger at the end ofthe run gently press it down until you feel it click When you remove your finger the plunger will pop up to its released position Plunger Released 2 Remove the rotor from the ultracentrifuge and place it in the rotor vise J Removethe 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 Refer to CHAPTER 3 for sample recovery methods 4 17 Using Rotors Vertical Tube and Near Vertical Tube Rotors TLR IM 9AC CHAPTER 5 Care and Maintenance Introduction This section provides information on the care of rotors and accessories Included is a list of some common operating problems with suggestions for their solutions Rotors and accessories should be kept in optimal condition to minimize the chance of rotor or labware failure In addition to these instructions observe procedures and precautions provided in individual rotor manuals Appendix A of this manual provides the chemical resistances of rotor and accessory materials to various acids bases salts and solvents Rotor Care Rotor care involves not only careful operating procedures bu
50. e for each rotor 4 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 Spacer _ 7 Tube Plug _ Tube 1 Leave buckets without tubes completely empty b IfQuick Seal tubes are being used install spacers and or floating spacers over sealed tubes refer to the applicable rotor manual g Max Metal Floating Spacer Spacer Y Dome Top Bell Top 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 5 Match numbered caps with numbered buckets a Screw the caps into the bucket until there is metal to metal contact 4 8 TLR IM 9AC Using Rotors 4 Swinging Bucket Rotors 6 Attach numbered buckets loaded or empty to corresponding rotor body positions Loaded buckets must be arranged symmetrically on 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 J NOTE Two tubes can be run if the filled buckets are attached in opposing positions on the rotor positions 1 and 3 or 2 and 4 and the two remaining buckets are also attached If you regularly run only two filled buckets
51. e of plastic Bleach B y P 8 p B sodium hypochlorite may be used but may cause discoloration of anodized surfaces Use the minimum immersion time for each solution per laboratory standards ww Inspection Frequent and thorough inspection is crucial to maintaining a rotor in good operating condition nU 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 t In U S contact Biodex Medical Systems Shirley New York internationally contact the U S office to find the dealer closest to you t Flammability hazard Do not use in or near operating ultracentrifuges TLR IM 9AC 5 3 5 Care and Maintenance Rotor Care 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 Ifany ofthese 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 Lid Assembly b w Threads Lid O ring outer RB m ra O ring s inner Check for Corrosion 2 Regularly check the condition of O rings and replace any that are worn or damaged 3 Re
52. e 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 Careful centrifugation technique is essential because forces generated in ultracentrifugation can be enormous For example 1 gram at the bottom of an TLA 100 3 rotor rotating at 100 000 rpm exerts the gravitational equivalent of over 0 5 ton of centrifugal mass at the bottom of the tube cavity Some of the newer rotors see Table 1 1 can be used in the TL 100 ultracentrifuge no longer manufactured only if the ultracentrifuge is updated with a new drive spindle and updated operating software modification kit number 360477 Operation of these rotors in an unmodified TL 100 may cause the rotor to stick or slip on the spindle NOTE Specific information about filling sealing and capping containers loading rotors etc can be found in later sections Rotor Balance TLR IM 9AC 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 Wei
53. ecd Mr DE SN E z ecu esee od e ect d s ees E E o lalele ax aja adal fit 2146 4 thinwall transparent yes yes no S U U M S U U U U U S polypropylene thickwall translucent no nob yes S S S M S M M U M U S polypropylene Ultra Clear transparent yes yes no S U U S U U U U U U M polycarbonate transparent no no yes M U U M U U U U U M M polypropylene translucent no no yes S S S M S M S M M 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 U unsatisfactory resistance S satisfactory resistance M marginal resistance a Refer to Appendix A for information about specific solutions b 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 Do not use flammable substances in or near operating centrifuges 2 2 TLR IM 9AC Tubes and Accessories 2 Labware Types Labware Material Compatibility with Solvents and Sample The chemical compatibility of tube 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 or polycarbonate tubes Polycarbonate and Ultra Clear tubes are inc
54. ecovering fractions from stem nare to Quick Seal tubes One of the following procedures may be provide an air inlet 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 ofthe 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 Sample out Aspirate the sample from the tube top by snipping off p the tube stem then aspirating the sample with a Pasteur pipette or needle and syringe gt Slice the tube using the Beckman CentriTube Slicer m i x 347960 and adapter 354526 H CentriTube Slicer E 347960 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 available at www beckmancoulter com 3 16 TLR IM 9AC Using Tubes and Accessories 3 Making Ultra Clear Tubes Wettable Making Ultra Clear Tubes Wettable TLR IM 9AC 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
55. ed with the individual rotor instruction manual shipped 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 tabletop 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 adapters and spacers to help you choose a particular tube for your application CHAPTER 3 provides instructions for using tubes and related accessories CHAPTER 4 contains step by step procedures for preparing each type of rotor for a centrifuge run CHAPTER 5 provides rotor tube and accessory care and maintenance information as well as some diagnostic hints Please read it Proper rotor c
56. eful when recovering sample from tubes Open Top Tubes The usual methods of recovering supernatants or pellets include decanting or withdrawing the gradient and scraping pellets from the tube bottom 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 OptiSeal Tubes Centrifugation exerts high forces on plastic labware The effect of these forces on OptiSeal labware is compression ofthe 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 leak 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 3 12 TLR IM 9AC Using Tubes and Accessories Sample Recovery After centrifugation use the spacer removal tool 338765 or a hemostat to carefully remove the spacers taking care not to scratch the rotor cavities Spacer Removal Tool A tube will sometimes come out of the rotor cavity along with the spacer a Separate the tube from the spacer with a twisting motion NOTE Centrifugation causes a slight vacuum to build up
57. eous solution will adhere to the more elastomers used in various accessories should wettable a tube or bottle material is the more be used at temperatures between 59 and 232 C biological material DNA protein cells and so 75 and 450 F forth will adhere to the walls 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 Beckman 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 1S 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 PEl 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 TLR IM 9AC Glossary 3 Glossary
58. etal threads lubricate them with Spinkote lubricant 306812 Failure to keep threads properly lubricated can result in stripped or galled threads and stuck rotor components Rotor plug gaskets a component of vertical tube and near vertical tube rotors do NOT require lubrication but should be checked cleaned and or replaced as required Tube and Accessory Care Cleaning TLR IM 9AC Proper care of tubes involves observing temperature fill volume and run speed limitations as well as careful cleaning and sterilization procedures Do not wash tubes in a commercial dishwasher detergents and temperatures are too harsh 1 Wash tubes 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 2 Polycarbonate tubes are vulnerable to attack by alkaline solutions and detergents so use a detergent with pH less than 9 such as Solution 555 a Donotusea brush with exposed metal scratches in polycarbonate will cause early failure 5 5 5 Care and Maintenance Tube and Accessory Care 3 Alcohol and acetone react unsatisfactorily with many tube and accessory materials Ifa solvent must be used to rinse dry or decontaminate these materials consult APPENDIX A to select an appropriate solvent 4 Donot dry tubes or accessories in an oven Labware should be air dried 5 optiSeal Quick Seal Ultra Clear and thinwall polypropylene tubes a
59. ffect seal integrity and thus containment f No longer manufactured g At speed TLR IM 9AC Rotors Rotor Selection 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 forces and or long run times The relative pelleting efficiency of each rotor is measured by its k factor clearing factor EQ 1 k In Tmax Tmin 19 o 3600 where is the angular velocity of the rotor in radians per second 2tRPM 60 or 0 10472 x rpm Tmax is the maximum radius and rq is the minimum radius After substitution EQ2 p Q533 x 10 In imax tin 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 10 seconds It is usual practice to use the standard sedimentation coefficient 520 0 sc dr dt 1 w2r where dr dt is the sedimentation velocity TLR IM 9AC 1 9 1 Rotors Rotor Selection based on sedimentation in water at 20 C Clearing factors can be calculated at speeds
60. g 30 Angle 12 TLA 100 100 000 38 9 345 30 0 20x0 2 mL TL TB 003 Fixed Angle 436 000 x g 30 Angle 7 TLV 100 100 000 35 7 30 2 246 8x2 0mL TL TB 007 H Vertical Tube 400 000 x g 0 Angle 9 t MLN 80 80 000 542 43 3 32 5 8x8 0mL TL TB 022 Near 390 000 x g Vertical 20 Tube 9 Angle MLA 80 80 000 61 9 45 7 29 5 8x80mL TL TB 024 Fixed Angle 444 000 x g 26 Angle 29 MLA 55 55 000 84 6 64 0 446 8x135mL TLTB 026 Fixed Angle 287 000 x g 35 Angle 53 1 Rotors Rotor Selection Table 1 1 Rotors Used in Beckman Coulter Tabletop Ultracentrifuges Continued Radial Distances Number of mm Tubes x Max Speed Nominal Rotor RCF Capacity Manual Rotor Profile and Description k factor Tmax fav min largest tube Number TLA 55e 55 000 55 0 480 25 0 12x1 5mL TL TB 020 NS Fixed Angle 186 000 x g J 45 Angle 66 7 TLS 55 55 000 76 5 59 4 422 4x22mL TL TB 006 d 1 Swinging 259 000 xg i Bucket 50 90 Angle9 MLS 50 50 000 958 71 11 475 4x5mL TL TB 023 Swinging 268 000 x g Bucket 71 90 Angle9 MLA 50 50 000 832 58 4 33 6 6x32 4mL B03896 Fixed Angle 233 000 x g 30 Angle 92 TLA 45f 45 000 55 0 48 0 25 0 12x1 5mL TL TB 012 S Fixed Angle 125 000 x g 45 Angle 99 1 8 a Maximum speeds are based on a solution de
61. g to each particle s buoyant density 2 Select the curve at the desired temperature 4 C that gives the best particle separation 3 Note the run speed along the selected curve 60 000 rpm 4 From Figure B 1 select the maximum homogeneous CsCl density in this case 1 63 g mL that corresponds to the temperature and run speed established above These parameters will provide the particle banding pattern selected in Step 2 In this example particles will band at about 37 3 and 41 0 mm from the axis of rotation about 3 7 mm apart When the tube is held upright there will be about 3 8 mm of center of band to center of band separation TLR IM 9AC B 3 The Use of Cesium Chloride Curves Typical Examples for Determining CsCl Run Parameters B 4 Figure B 1 Precipitation Curves for the MLN 80 Rotor Homogeneous CsCl Solution g mL 1 90 1 85 1 80 1 75 RN AN 1 70 LA ar 1 65 p ge SY S 1 60 MLN 80 ROTOR 1 55 20 C 4 C 1 50 1 45 1 40 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70
62. ght of opposing 1 13 1 Rotors General Operating Information 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 e O D cod 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 distribute the sample symmetrically e Use thickwall tubes partially filled to distribute sample to additional tubes Layeralow 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 1 14 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
63. ght tubes can be prepared for use at once in the specially designed racks listed in Table 3 2 1 Make sure that no fluid is in the tube stem and that the stem is clean and dry 2 Inserta Noryl plug assembly plug and O ring shipped assembled in each tube stem z 3 5 Using Tubes and Accessories Filling and Sealing Quick Seal Tubes J Set the plug seating bar on the rack ensuring that the pegs at each end fit into the rack openings 4 Press firmly straight down all along the top of the bar When you remove the bar the plugs should be straight and seated into the stems 5 Check the tubes to be sure all plugs are seated a Ifany plugs are not seated seat them individually i ss O ring AN No fluid appeals above O ring as wide black line Filling and Sealing Quick Seal Tubes Fill each tube to the base of the neck using a syringe with a 13 gauge or smaller needle A small air space no larger than 3 mm may be left but an air bubble that is too large can cause the tube to deform disrupting gradients or sample Spacer and or floating spacer 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 Tub
64. gularly check that all sealing surfaces are smooth and undamaged to ensure proper sealing 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 _ Rotor Plug _ Gasket Check z for Corrosion 3 5 4 TLR IM 9AC Care and Maintenance Tube and Accessory Care Field Rotor Inspection Program The Field Rotor Inspection Program FRIP has two purposes 1 to prevent premature rotor failures by detecting conditions such as stress corrosion metal fatigue damage or wear in the anodized coatings and 2 toinstruct laboratory personnel in the proper care of rotors Beckman Coulter has trained a group of experienced service engineers in the techniques of nondestructive evaluation For more information about the program contact your Beckman Coulter representative Lubrication Proper lubrication is essential to obtain specified torque values where required and to minimize thread wear Many rotors use O rings as seals to maintain atmospheric pressure in the rotor during a run These O rings and the surfaces they bear against must be kept clean and evenly lubricated After removing and cleaning rotor O rings or gaskets lightly but evenly coat them with silicone vacuum grease 335148 and reposition them in the rotor After cleaning m
65. hese 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 TLR IM 9AC 4 11 Using Rotors Vertical Tube and Near Vertical Tube Rotors Figure 4 3 Vertical Tube Rotor min__ lav Tmax TLV 100 Axis of Rotation Near Vertical Tube Rotors Description Tubes in near vertical tube rotors see Figure 4 4 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 45 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 Figure 4 4 Near Vertical Tube Rotor min lay seu Tmax TLN 100 Axis of Rotation Tubes 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 manual
66. ide 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 B of this manual 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 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 Consult individual rotor manuals for allowable run speeds TLR IM 9AC 1 15 1 Rotors General Operating Information 1 16 TLR IM 9AC CHAPTER 2 Tubes and Accessories Introduction This section describes various labware used in Beckman Coulter ML and TL series rotors General instructions for using containers follow in CHAPTER 3 Care and maintenance instructions are in CHAPTER 5 General rotor use instructions are in CHAPTER 4 The individual rotor manual that comes with each rotor provides specific instructions on the tubes 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 design or material meets all application requirements Labware choice is usually b
67. iercing the tube to avoid pushing the needle out the opposite side b Puncture the bottom of the tube and collect the drops Ew 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 347960 and CentriTube Slicer Adapter 354526 Tubes are pressurized after centrifugation so pierce the tube top with a needle to relieve pressure before slicing CentriTube Slicer 347960 3 14 TLR IM 9AC Using Tubes and Accessories 3 Sample Recovery Removing Plugs from Tables 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 hold 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 J while pressing down on the rack insert use the extraction tool to firmly grasp the plug 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 D Repeat for each tube a TLR IM 9AC 3 15 Using Tubes and Accessories Sample Recovery Quick Seal Tubes 1 lt Cut Quick Seal There are several methods of r
68. ing and Sealing or Capping Requirements 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 Table 3 1 Filling and Capping Requirements for Tubes Tube or Bottle Polypropylene thinwall tubes thickwall tubes OptiSeal tubes Quick Seal tubes konical Quick Seal tubes konical open top tubes Swinging Bucket Rotors within 2 3 mm of top at least 1 2 full full and plugged full and heat sealed full and heat sealed within 2 3 mm of top Filling Level Requirements Fixed Angle Rotors full and capped 1 2 full to max capless level full and plugged full and heat sealed Vertical and Near Vertical Tube Rotors full
69. irements Observe the maximum rotor speeds and fill volumes listed in the rotor manual Rotor Preparation and Loading 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 Prerun Safety Checks Read all safety information in the rotor manual before using the rotor 1 Make sure that the rotor body buckets and bucket caps are clean and show no signs of corrosion or cracking 2 check the chemical compatibilities of all materials used Refer to APPENDIX A Verify that tubes and accessories being used are listed in the applicable rotor manual Rotor Preparation and Loading 1 Be sure that bucket threads are clean and lightly but evenly lubricated with Spinkote lubricant 306812 as required 2 Remove the bucket O rings and coat them lightly but evenly with silicone vacuum grease 335148 a Install O rings in the buckets Never run a filled bucket without an O ring as the bucket contents may be lost leading to rotor imbalance and possible failure TLR IM 9AC 4 7 Using Rotors Swinging Bucket Rotors 3 Dry the exterior of the tubes 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 availabl
70. laboratory detergents are too harsh for aluminum rotors and components The Rotor Cleaning Kit 339558 contains two quarts of Solution 555 and brushes that will not scratch rotor surfaces 2 Rinse thoroughly with water J 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 5 Frequently clean all surfaces that contact O rings a Regularly clean the threads of the rotor lid plugs 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 Threads E 20 5 2 TLR IM 9AC Care and Maintenance Rotor Care Decontamination 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 Ifarotor 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 NO
71. man Coulter Field Service if a problem cannot be corrected NOTE Use only the labware listed in the applicable rotor manual Table 5 2 Troubleshooting Chart Symptom Possible Cause and Suggested Action Rotors Severe vibration Rotor imbalance To balance the rotor load fill all opposing tubes to the same level with liquid of the same density Weight of opposing tubes must be distributed equally Place tubes in a fixed angle near vertical tube or vertical tube rotor symmetrically as illustrated in CHAPTER 1 Figure 1 5 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 4 Stripped rotor plugs on Rotor vise not used wrong tool used incorrect torque or insufficient vertical tube or near pressure on plug adapter when tightening rotor plugs Observe careful vertical tube rotors tightening procedures Rotor lid or bucket cap Threads contaminated with dirt dried lubricant or metal particles is difficult to remove or threads insufficiently lubricated cause rotor components to stick after centrifugation Do not use excessive force to loosen components Contact Beckman Coulter Field Service Routinely clean metal threads with concentrated Solution 555 339555 then lubricate them with Spinkote 306812 Paint coming off where Not an ope
72. man Coulter representative who will inspect all of your rotors for corrosion or damage The representative will recommend repair or replacement of at TLR IM 9AC Safety Notice Mechanical Safety 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 TLR IM 9AC V vi Safety Notice Mechanical Safety TLR IM 9AC Contents _ Safety Notice iii Scope xiii CHAPTER 1 Rotors 1 1 Introduction 1 1 General Description 1 1 Rotor Designations 1 1 Material 1 2 Rotor Retention 1 3 Rotor Selection 1 3 Pelleting Differential Separation 1 9 Isopycnic Separations 1 12 Rate Zonal Separations 1 12 General Operating Information 1 13 Rotor Balance 1 13 Overspeed Protection 1 14 Allowable Run Speeds 1 15 CHAPTER 2 Tubes and Accessories 2 1 Introduction 2 1 Labware Selection Criteria 2 1 Labware Material Compatibility with Solvents and Sample 2 3 Gradient Formation and Fractionation 2 3 Labware Types 2 3 Polypropylene Tubes 2 3 Open Top Polypropylene Tubes 2 4 OptiSeal Tubes 2 4 Quick Seal Tubes 2 4 Polycarbonate Tubes 2 5 Polypropylene Tubes 2 5 Polyethylene Tubes 2 5 Ultra Clear Tubes 2 5 Cellulose Propionate Tubes 2 6 konical Tubes 2 6 vii Contents CHAPTER 3 CHAPTE
73. 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 TLR IM 9AC 3 9 Using Tubes and Accessories Filling and 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 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
74. nd gaskets in rotor assemblies should be used at temperatures between 34 and 121 C 30 and 250 F Centrifugal effect Accumulated value of 12 o dt t1 where t is time and w 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 pelleting efficiency In Tmax Tmin 10 s o 3600 or _ 253303 x In rg nin k RPM 1000 TLR IM 9AC 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 u
75. 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 Inspection TLR IM 9AC Inspect containers and accessories before use Inspect tubes for cracks or any major deformities before using them Donotuseatube that has become yellowed or brittle with age or excess exposure to ultraviolet light Crazing the appearance of fine cracks on tubes is the result of stress relaxation If a crack approaches the outer wall of the tube discard it Discard any deformed or cracked adapters ih Crazing Cracking 5 7 5 Care and Maintenance Returning a Rotor or Accessory to the Factory Tube and Bottle Storage Tubes have an indefinite shelf life if properly stored Store in a dark cool dry place away from ozone chemical fumes and ultraviolet light sources Removing Jammed or Collapsed Tubes Centrifugal force may collapse improperly filled or sealed 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
76. nsity of 1 7 g mL for all rotors except the MLA 80 solution density for the MLA 80 is 1 2 g mL The k factors are calculated for all Beckman Coulter rotors using the largest volume tube as a measure of the rotor s relative pelleting efficiency in water at 20 C Relative Centrifugal Field RCF is the ratio of the centrifugal acceleration at a specified radius and speed rw2 to the standard acceleration of gravity g according to the following formula RCF rw2 g where r is the radius in millimeters w is the angular velocity in radians per second 2 p RPM 60 and g is the standard acceleration of gravity 9807 mm s2 After substitution RCF 1 12 r RPM 1000 2 b Use only in the Optima MAX XP ultracentrifuge c Use only in Optima MAX MAX XP or MAX E ultracentrifuges d Before these rotors can be used in a TL 100 ultracentrifuge the instrument must be updated with a new drive spindle and updated operating software modification kit number 360477 Operation of these rotors in an unmodified TL 100 may cause the rotor to stick or slip on the spindle e This rotor was tested to demonstrate containment of microbiological aerosols under normal operating conditions of the associated Beckman Coulter centrifuge when used and maintained as instructed Validation of microbiological containment was done at an independent third party testing facility CAMR Porton Down UK or USAMRIID Ft Detrick MD U S A Improper use or maintenance may a
77. od 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 angle rotors Ultra Clear tubes are designed to be used one time only These tubes have good resistance to most weak acids and some weak bases but are unsatisfactory for DMSO and most organic solvents including all alcohols Ultra Clear tubes should not be autoclaved TLR IM 9AC 2 5 2 Tubes and Accessories Temperature Limits Cellulose Propionate Tubes Cellulose propionate tubes used in some fixed angle rotors are transparent and designed for one time use They are used without caps and should be full for centrifuging They should not be autoclaved or sterilized with alcohol These tubes have good tolerance to all gradient media including alkalines They are unsatisfactory for most acids and alcohols konical Tubes Cellulose propionate tubes used in some fixed angle rotors are transparent and designed for one time use They are used without
78. om the vise TLR IM 9AC 4 15 4 Using Rotors Vertical Tube and Near Vertical Tube Rotors Operation For runs at other than room temperature refrigerate or warm the rotor beforehand for fast equilibration Installing the Rotor Use an absorbent towel to wipe off condensation from the rotor then carefully place the rotor on the drive hub 2 TLseries rotors lock the rotor in place by gently pressing the plunger down until you feel it click When you remove your finger the plunger will remain flush with the rotor body if it is properly engaged a Ifthe plunger pops up repeat the procedure Plunger Locked The Optima MAX XP MAX TL MAX and MAX E ultracentrifuges automatically secure the rotor to the drive shaft without the need for engaging the plunger In all tabletop ultracentrifuge models except the Optima MAX XP MAX TL MAX and MAX E it is very important to lock the rotor in place before beginning the run to ensure that the rotor remains seated during centrifugation Failure to lock the rotor in place before beginning the run may result in damage to both rotor and instrument 3 Refer to the centrifuge instruction manual for detailed operating information N 4 16 TLR IM 9AC TLR IM 9AC Using Rotors 4 Vertical Tube and Near Vertical Tube Rotors Removal and Sample Recovery If disassembly reveals evidence of leakage you should assume that some fluid escaped the rotor Apply appropriat
79. ompatible with DMSO sometimes used in the preparation of sucrose gradients for sedimentation of denatured DNA Refer to APPENDIX A for detailed compatibility information 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 tube 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 T bes 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 Pol
80. ontrary to the instructions in the operator s manual s for the Beckman Coulter Centrifuge System components in use This includes but is not limited to operator misuse abuse or negligence regarding indicated maintenance procedures centrifuge and rotor classification requirements proper speed reduction for the high density of certain fluids tubes and tube caps speed reduction for 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
81. or use in certain rotors Nul E Plug 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 Tubes Meta Spacer Ly Dome Top 2 4 g Max Heat sealed Quick Seal tubes are used in swinging bucket vertical Floating Spacer iima Quick Seal tubes are a convenient form of sealable tube they are lil especially useful for the containment of radioactive or pathogenic tube near vertical tube and in most fixed angle rotors Single use samples There are two Quick Seal tube designs dome top and bell top The bell top simplifies removal of materials that float during centrifugation Dome top tubes hold more volume than their bell top equivalents Bell Top Detailed information about Quick Seal tubes is contained in publication IN 181 TLR IM 9AC Tubes and Accessories Labware Types Polycarbonate Tubes Polycarbonate is tough rigid nonwettable and glass like in appearanc
82. otor type and serial number history of use approximate frequency of use reason for the return original purchase order number billing number and shipping number if possible name and email address of the person to be notified upon receipt of the rotor or accessory at the factory and name and email address of the person to be notified about repair costs etc TLR IM 9AC Care and Maintenance 5 Diagnostic Hints To protect our personnel it is the customer s responsibility to ensure that the parts are free from pathogens chemical 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 TLR IM 9AC Some of the more common operating problems experienced in centrifugation are listed in Table 5 2 with suggestions for their solutions Contact Beck
83. r Solvents Maximum Density Materials Solvent at 20 C Sucrose 66 H20 1 32 Sucrose 65 D O 1 37 Silica sols H20 1 30 Diodon HO 1 37 Glycerol 1 26 H20 Cesium chloride H20 1 91 D0 1 98 Cesium formate H20 2 10 Cesium acetate H20 2 00 Rubidium chloride 1 49 XL H20 Rubidium formate 1 85 H 0 Rubidium bromide H20 1 63 Potassium acetate H20 1 41 potassium formate H0 1 57 D O 1 63 Sodium formate 1 32 H20 1 40 Lithium bromide pom 1 83 H20 Lithium chloride D20 1 33 Albumin H20 1 35 Sorbitol H20 1 39 Ficoll H2O 1 17 Metrizamide 1 46 H20 TLR IM 9AC Gradient Materials Introduction Table C 2 Density Refractive Index and Concentration Data Cesium Chloride at 25 C Molecular Weight 168 37 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 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 37
84. r 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 2 6 TLR IM 9AC Tubes and Accessories Adapters angle rotors the top of the tube must be supported In near vertical tube and vertical tube rotors the entire tube cavity must be filled Spacer Floating Spacer 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 ofthe 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 Adapters Many rotors can accommodate a variety of tube sizes by using adapters that line the tube cavity or bucket Small open top tubes use Delrin adapters which line the tube cavity or bucket Adapters with conical cavities must be used to support both open top and Quick Seal konical tubes AM Adapters Tubes used with adapters can be filled and capped according to
85. ration may involve the use of solutions and test samples that are pathogenic toxic or radioactive Such materials should not be used in these rotors however unless all necessary safety precautions are taken Observe all cautionary information printed on the original solution containers prior to their use Handle body fluids with care because they can transmit disease No known test offers complete assurance that 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 acces
86. rational problem bucket contacts rotor pocket on swinging bucket rotor 5 9 Care and Maintenance Diagnostic Hints 5 10 Table 5 2 Troubleshooting Chart Continued Symptom Possible Cause and Suggested Action Tubes T be 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 CHAPTER 3 for instructions on filling and plugging OptiSeal tubes Quick Seal tubes Improperly sealed After heat sealing squeeze the tube gently if the tube contents may be disturbed to test the seal for leaks If the tube leaks reseal it Tube cracking 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 Tubes may become brittle with age and use Dispose of brittle or cracked tubes Tube collapse Thinwall tube volume too low to provide tube wall support Meniscus should be 2 to 3 mm below the tube top Referto the rotor manual for tube volumes Moisture between the tube and the cavity or bucket can cause the tube
87. re intended for one time use and should be discarded after use Decontamination ee 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 material Sterilization and Disinfection Refer to Table 5 1 for sterilization methods recommended for each container type Most tubes and accessories except those made of Ultra Clear polyethylene cellulose propionate or Noryl 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 should be placed open end down or supported in a rack if autoclaved Do not autoclave plastic adapters or spacers Do not autoclave sealed or capped tubes Pressure in a sealed container can cause an explosion Pressures within the autoclave can cause partially sealed containers to collapse when the autoclave vents 121 C EN C amm A cold sterilization method such as immersion in 1096 hydrogen peroxide for 30 minutes may be used on Ultra Clear tubes Refer to Table 5 1 to select cold sterilization materials that will not damage tubes and accessories 5 6 TLR IM 9AC Care and Maintenance Tube and Accessory Care Table 5
88. sects the homogeneous CsCl density 70 000 rpm In Figure B 2 sketch in a horizontal line corresponding to each particle s buoyant density Mark the point in the figure where each particle density intersects the curve corresponding to the selected run speed and temperature Particles will band at these locations across the tube diameter at equilibrium during centrifugation In this example particles will band about 44 3 and 46 6 mm from the axis of rotation about 2 3 mm of centerband to centerband separation at the rotor s 9 degree tube angle When the tube is held upright Gradients in Figure B 2 result from homogeneous CsCl solutions but can be more rapidly generated from step or linear gradients as long as the total amount of CsCI in solution is equal to the amount in the homogeneous solution from the curves in Figure B 1 TLR IM 9AC The Use of Cesium Chloride Curves B Typical Examples for Determining CsCl Run Parameters there will be about 2 4 mm of centerband to centerband separation This interband distance d can be calculated from the formula 2 dp COSg where dg is the interband distance when the tube is held at an angle 9 in the rotor At Speed At Rest Floating in Rotor upright Components EN Bands Pelleted Material Example B Knowing particle buoyant densities for example 1 59 and 1 54 g mL how do you achieve good separation 1 InFigure B 2 sketch in a horizontal line correspondin
89. sed 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 volumes 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 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
90. sed on differential rate of sedimentation using a preformed gradient with the sample layered as a zone on top of the gradient RCF Relative centrifugal field the ratio of the centrifugal acceleration at a specified radius and speed ra 2 to the standard acceleration of gravity g according to the following equation ro RCF 72 g where r is the radius in millimeters is the angular velocity in radians per second 21 RPM 60 and g is the standard acceleration of gravity 9807 mm s2 Thus the relationship between RCF and RPM is 2 M RCF 1 12 RPM T T000 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 Fmin 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 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 TLR IM 9AC Glossary Sedimentation coefficient s Sedimentation Viton Fluorocarbon elastomer used in high velocity per unit of centrifugal force temperature applications Viton is a registered trademark of E l Du Pont de Nemours amp s dE els Company dt dg Wettable Tube or bottle material that water or Silicone rubber A large group of silicone other aqu
91. 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 reduced maximum speed rated speed IER B 1 where p is the density of the tube contents This speed reduction will protect the rotor from excessive stresses due to the added tube load Note however that the use of this formula may still produce maximum speed figures that are higher than the limitations imposed by the use of certain tubes or adapters In such cases use the lower of the two figures 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 B 1 ensure that CsCl will not precipitate in the MLN 80 rotor Curves are provided at two temperatures 20 C black lines and 4 C gray lines Note from Figure B 1 that for a given CsCl density faster rotor speeds can be used as the fill volume in the tube decreases from full to one quarter filled Also for a given rotor speed the maximum CsCl density that can be s
92. side tube contents will not spill but make sure the tube does not roll After 2 hours at room temperature slowly set the tube upright 1 mL Syringe __ 20 to 22 Gauge Needle g n 2 to 3mm 45 to 50 Gradient Once the gradient is prepared layer the sample on top of the gradient 2103 mm 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 Buffer Sample NOTE Ifa partially filled thickwall tube is centrifuged the tube does not with MN S i 2 to 3 require liquid support and therefore the buffer solution is not required Sucrose Added Gradient Cesium Chloride Gradients Cesium chloride gradients can be made by filling the tube with a homogeneous solution of CsCl and sample Select a homogeneous CsCl solution density so that when it is distributed its density range will encompass the density of the particle s of interest Refer to APPENDIX B for an explanation of the use of the CsCl curves General Filling and Sealing or Capping Requirements See Table 3 1 for general filling and sealing requirements for tubes used in ML or TL series preparative rotors Maximum fill volume includes sample and gradient Refer to individual rotor manuals for specific filling and capping requirements 3 2 TLR IM 9AC TLR IM 9AC Using Tubes and Accessories General Fill
93. sories Mechanical Safety Use only the rotors components and accessories designed for use in the rotor and centrifuge 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 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 Program FRIP This program involves a visit to your laboratory by a specially trained Beck
94. structions 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 Its density range should be sufficient to permit separation of the particles of interest by the chosen density gradient technique without overstressing the rotor Itshould not affect the biological activity of the sample It should be neither hyperosmotic or hypoosmotic when the sample is composed of sensitive organelles It should not interfere with the assay technique Itshould be removable from the purified product It should not absorb in the ultraviolet or visible range Itshouldbe inexpensive and readily available more expensive materials should be recoverable for reuse Itshould be sterilizable 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 TLR IM 9AC C 1 Gradient Materials Introduction C 2 Table C 1 Commonly Used Gradient Materials with Thei
95. t also careful attention to Regular cleaning decontamination and or sterilization as required Frequent inspection Corrosion prevention and Regular and proper lubrication Do not use sharp tools 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 Cleaning Wash rotors and rotor components immediately if salts or other corrosive materials are used or if spillage has occurred a DO NOT allow corrosive materials to dry on the rotor otor Cleaning Kit 339558 ae 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 M TLR IM 9AC 5 1 Care and Maintenance Rotor Care 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
96. 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 Titanium rotors are stronger and more chemical resistant than the aluminum rotors Exterior surfaces of titanium rotors are finished with black polyurethane paint 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 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 TLR IM 9AC Rotors 1 Rotor Selection coated with silicone vacuum grease Plug gaskets in vertical tube or near vertical tube rotors are made of Hytrel and do not require coating Rotor Retention A rotor retention mechanism on the ultracentrifuge drive hub secures the rotor during the run A plunger mechanism in the rotor is used to secure a TL series rotor to the drive hub before the run begins see Figure 1 2 Engaging the plunger ensures that the rotor does not slip on the h
97. the TLA 120 3 make it possible to distinguish between different rotors that have the same maximum allowable speed An example of each rotor type is shown in Figure 1 1 Tubes in fixed angle rotors designated MLA or TLA are held at an angle to the axis of rotation in numbered tube cavities The bodies of some rotors are fluted to eliminate unnecessary weight and minimize stresses In swinging bucket rotors designated MLS or TLS containers are held in rotor buckets 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 TLV 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 Material Figure 1 1 Fixed Angle Swinging Bucket Vertical Tube and Near Vertical Tube Rotors Vertical Tube Rotor Near Vertical Tube Rotor Tubes in near vertical tube rotors designated MLN or TLN are also held at an angle to the axis of rotation in numbered tube cavities However the reduced tube angle of these rotors typically 7 to 10 degrees reduces run times from fixed angle rotors with tube angles of 20 to 45 degrees while allowing components
98. 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 Delrin is a registered trademark of E Du Pont de Nemours amp Company TLR IM 9AC 2 7 Tubes and Accessories Adapters 2 8 TLR IM 9AC CHAPTER 3 Using Tubes and Accessories Em Introduction This section contains general instructions for filling and capping the labware used in Beckman Coulter preparative rotors for selecting and using the appropriate accessories and for recovering samples after a run Individual rotor manuals provide specific instructions on tubes and accessories that can be used in a particular rotor Rotor use instructions are in CHAPTER 4 A table of chemical resistances is in APPENDIX A of this manual Reference information on some commonly used gradient materials is in APPENDIX C Gradient Preparation Many commercial gradient formers are available These devices usually load a tube Added T by allowing the gradient solutions to run down the side of the tube The heaviest First concentration is loaded first followed by successively lighter concentrations This method is acceptable for wettable tubes however loading a nonwettable tube such as Ultra Clear polypropylene and polycarbonat
99. tor 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 1 12 Particle separation achieved with rate zonal separation is a function of the particles sedimentation coefficient density size and shape and viscosity of the gradient material Sucrose is especially useful as a gradient material for rate zonal separation because its physical characteristics are well known and it is readily available Samples are layered on top of the gradient Under centrifugal force particles migrate as zones Rate zonal separation is time dependent if the particles are more dense than the most dense portion ofthe 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 ro
100. tors 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 kt factor and are given in the applicable rotor manuals These constants can be used to estimate the time TLR IM 9AC Rotors General Operating Information t in hours required to move a zone of particles of known sedimentation coefficient and density to the bottom of a 5 to 2096 gradient EQ 7 ll v 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 c 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 time t Conversely if the value of t is known sedimentation coefficients can be estimated from zone positions In most cases when banding two or three components by rate zonal separation run times can be considerably reduced by using reduced fill levels Tubes are partially filled with gradient but the sample volume is not changed however gradient capacity will be reduced Thickwall tubes should be used when this technique is employed since thinwall tubes will collapse if not full If swinging bucket rotors are used with preformed shallow gradients 5 to 2096 or if fixed angle vertical tube or near vertical tube rotors ar
101. ub during initial acceleration and that it remains seated during centrifugation The Optima MAX XP MAX TL MAX and MAX E ultracentrifuges automatically secure the rotor to the drive shaft without the need for engaging the plunger Figure 1 2 Plunger Mechanism in Locked and Released Positions Plunger Locked Plunger Released In all tabletop ultracentrifuge models except the Optima MAX XP MAX TL MAX and MAX E it is very important to lock the rotor in place before beginning the run to ensure that the rotor remains seated during centrifugation Failure to lock the rotor in place before beginning the run may result in damage to both rotor and instrument Rotor Selection Selection of a rotor depends on a variety of conditions such as sample volume number of sample components to be separated particle size run time required 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 Refer to CHAPTER 4 for specific information about the use of each type of rotor Vertical tube rotor shown TLR IM 9AC 1 3 Rotors Rotor Selection ie m CU VU Fixed angle rotors are general purpose rotors that are especially useful for pelleting s
102. ubcellular 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 3 compared to swinging bucket rotors resulting in reduced run times Swinging bucket rotors are used for 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 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 3 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 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
103. ube TLR IM 9AC 3 7 Using Tubes 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 a Do not scratch the interior of the formers as you may damage this coating Seal Former J Seal each tube using Method A With the Seal Guide or B Without the Seal Guide 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 Teflon is a registered trademark of E I Du Pont de Nemours amp Co 3 8 TLR IM 9AC Using Tubes and Accessories 3 Filling and Sealing Quick Seal Tubes Method A With the Seal Guide Seal Guide 1 Place a seal guide with the flat side down over the seal former 2 Turn the Tube Topper pushbutton to USE position Press the pushbutton and wait 3 to 5 seconds for the tip to heat 3 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
104. y 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 Use the required spacers and or floating spacers if necessary to complete the loading operation a IfOptiSeal tubes are being used install a spacer over each plugged tube refer to the applicable rotor manual a Spacer Tube Plug _7 Tube 1 Leave cavities without tubes completely empty 4 3 4 Using Rotors Fixed Angle Rotors b IfQuick Seal tubes are being used install spacers and or floating spacers over sealed tubes refer to the applicable rotor manual Metal Spacers NI Floating I Spacer a Dome Top Bell Top Tube LC Tube 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 1 Leave cavities without tubes completely empty 4 After the rotor is loaded insert it into the portable polypropylene rotor vise 346133 a Placethe lid on the rotor and tighten it firmly to the right clockwise by hand Notoolis required Operation For runs at other than room temper
105. ypropylene 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 TLR IM 9AC 2 3 Tubes and Accessories Labware Types Open Top Polypropylene Tubes Thinwall open top tubes are used in swinging bucket and fixed angle rotors In swinging bucket rotors thinwall tubes should be filled to within 2 or 3 mm of the tube top for proper tube support Caps are usually required in fixed angle rotors Thinwall tubes are designed for one time use and should be discarded after use Thickwall open top tubes offer the convenience of centrifuging partially filled tubes without tube caps in fixed angle and swinging bucket rotors Because the solution re orients during centrifugation the maximum partial fill volume depends on the tube angle For greater fill volumes use tubes with caps Refer to J the applicable rotor manual for fill volumes and speed reduction requirements Thickwall polypropylene tubes are typically reusable unless deformed during centrifugation or autoclaving OptiSeal Tubes Spacer OptiSeal tubes single use tubes designed f

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