User Guidelines & Standard Operating Procedure for the Beckman Avanti J-30I High Performance Centrifuge
Centrifuge Standard Operating Procedure ii TABLE OF CONTENTS DISCLAIMER ................................................................................ iv ACKNOWLEDGEMENTS .....................................................................v 1. INTRODUCTION........................................................................1 1.1 Purpose of the Standard Operating Procedure .............................1 1.2 Centrifugation Theory ..........................................................1 1.2.
Centrifuge Standard Operating Procedure iii APPENDIX 1: ARABIDOPSIS CHLOROPLAST ISOLATION AND CHLOROPHYLL DETERMINATION.......................................................................... 22 APPENDIX 2: CENTRIFUGE USER LOG.................................................. 25 APPENDIX 3: PREVENTATIVE MAINTENANCE LOG ....................................
Centrifuge Standard Operating Procedure iv DISCLAIMER The materials contained in this document have been compiled from sources believed to be reliable and to represent the best opinions on the subject. This document is intended to serve only as a starting point for good practices and does not purport to specify minimal legal standards.
v Centrifuge Standard Operating Procedure ACKNOWLEDGEMENTS The following individuals of Laurier contributed to the writing, editing, and production of this manual: Gena Braun (Instrumentation Technician); Stephanie Kibbee (Environmental/Occupational Health and Safety Office); Arthur Szabo (Chemistry). This manual was prepared for Laurier. Any corrections, additions or comments should be brought to the attention of the Instrumentation Technician at 519-884-0710 ext. 2361.
Centrifuge Standard Operating Procedure 1 1. INTRODUCTION 1.1 Purpose of the Standard Operating Procedure This standard operating procedure (SOP) is NOT a substitute for training and/or reading the appropriate manuals before use. All principle investigators and supervisors must document that training has been received by students and staff who will be using the centrifuge. A list of authorized users will be kept by the Instrumentation Technician in SR314A.
2 Centrifuge Standard Operating Procedure T1 T2 k1 k 2 When using a specific rotor, you can determine how quickly a given particle will pellet by using the sedimentation coefficient (S) for that particle. The Svalue is expressed in Svedberg units and a larger S-value indicates faster sedimentation. The time taken to pellet a given particle can be determined by: T k S where T= pellet time in hours. k = pelleting efficiency of the rotor, and S = sedimentation coefficient of the particle. 1.2.
3 Centrifuge Standard Operating Procedure Table 1-1: Media suitability for density gradient separations applications (Basics of Centrifugation, Cole Parmer Technical Library) Gradient Media Cells Viruses Organelles Nucleoproteins Macromolecules Sugars Limited use Good Good Limited Use Not suitable Polysaccharides (e.g. Ficoll) Good for some applications Good for some applications Good for some applications Not suitable Not suitable Colloidal Silica (e.g.
4 Centrifuge Standard Operating Procedure Table 1-2: Rotor suitability for various applications applications (Basics of Centrifugation, Cole Parmer Technical Library) Type of rotor Type of Separation Pelleting Rate-zonal Sedimentation Isopycnic Good for macromolecules Fixed Angle Excellent Limited Swinging Bucket Inefficient Good Vertical Not suitable Good Poor for cells and organelles Good for cells and organelles Excellent Compatible Tube Types Thick wall open top and thin wall sealed Thic
5 Centrifuge Standard Operating Procedure Table 1-3: Rotors available for the Avanti J-30I centrifuge Rotor Maximum RPM and RCF (Critical Speed Range) k 30,000 RPM 108,860 x g JA 30.50 Ti Fixed 34o Angle 280 Tube Dimensions, Number of Tubes x Maximum Tube Volume Tubes: 25 X 105 mm Samples: 8 x 40 mL (600-800 RPM) Suitable Applications Pelleting of cells, cell particles, and subcellular fractions. 10,000 RPM 18,600 x g JLA 10.500 Fixed 20o Angle (600- 800 RPM) 13,000 rpm 26,500 x g JS 13.
6 Centrifuge Standard Operating Procedure Table 1-4: Tubes that can be used in each rotor1 Rotor Tube JA 30.50 JS 13.1 JA 10.
Centrifuge Standard Operating Procedure 7 2. POTENTIAL HAZARDS NEVER CENTRIFUGE MATERIALS THAT ARE CAPABLE OF DEVELOPING FLAMMABLE OR EXPLOSIVE VAPOURS. DO NOT CENTRIFUGE RADIOACTIVE, PATHOGENIC, OR TOXIC SUBSTANCES IN THIS CENTRIFUGE. Centrifuges have the capacity to be VERY dangerous, and care must be used each time a run is set up. Do not bump, lean on, or attempt to move the ultracentrifuge while it is running.
8 Centrifuge Standard Operating Procedure When running biological samples (not pathogens or toxic substances, as stated above), use sealed or capped tubes to minimize aerosol generation. In the event of a power failure, contact the Instrumentation Technician for assistance to retrieve samples from the ultracentrifuge. 1 1 1 2 1 1 2 2 1 1 1 3 3 1 2 Figure 2-1: Methods for balancing a rotor. In this example, if three tubes are placed in the rotor they must all be the same weight. 3.
Centrifuge Standard Operating Procedure 9 4. SPILL AND ACCIDENT PROCEDURES 4.1 Accidents If you notice anything unusual concerning centrifuge operation (smells, noises, etc.) stop the centrifuge immediately and contact the Instrumentation Technician. All incidents must be reported to the Instrumentation Technician and if applicable, a student’s supervisor.
Centrifuge Standard Operating Procedure 10 Health and Safety Office is also available to provide guidance at ext. 2874. The guidelines below are summarized from the WLU Laboratory Health and Safety Manual. Determine if the spill is a major or minor spill (see Table 4-1). 1) For major spills: a) Evacuate the lab, close the doors, restrict the area, and notify others in the area of spill, including your supervisor and the Instrumentation Technician if possible.
11 Centrifuge Standard Operating Procedure Table 4-1: Guidelines for classification of a major spill Material Air and water reactive materials Flammable liquids Combustible liquids Non-flammable organic liquids Concentrated acids Concentrated bases and alkalis Mercury Oxidizers Highly toxic, highly malodorous material Low hazard material Compressed gas leaks Quantity All quantities Greater than 4L Greater than 4L Greater than 4L Liquids greater than 1L Solids greater than 1kg Liquids greater than 1L Soli
Centrifuge Standard Operating Procedure 12 6. PROTOCOL 6.1 Sample Preparation 1. The rotors for the Beckman Avanti J30-I are stored in the cold room (SR412A). 2. Check the rotor before using: a. Make sure that it is clean and dry. If it is not clean, you must clean it before using it and report the problem to the Instrumentation Technician. Liquids and dried samples that are left in the buckets can cause corrosion and make the rotor unbalanced. b.
13 Centrifuge Standard Operating Procedure 6.2 Running the Centrifuge 1. Check the log book first to ensure that the centrifuge is functioning properly (i.e. no serious problems are listed). 2. Turn on the power switch; 3. Use the foot pedal at the front of the centrifuge to open the centrifuge lid; 4. If using the JA 30.50 rotor: a.
Centrifuge Standard Operating Procedure 14 the drive spindle hub. There are arrows on the rotor to direct placement of the rotor on the spindle. d. When the yoke is correctly seated, secure it to the drive spindle hub by hand tightening the tie-down knob. If the rotor is left in the centrifuge between runs, tighten the knob before each run. e. Close the centrifuge door only when you are sure the samples and rotor have been correctly balanced and attached to the centrifuge; f.
Centrifuge Standard Operating Procedure 15 0 RPM can be programmed for 1-10 minutes), or “OFF” (no brake is used, and the rotor can take up to 1 hour to coast to a stop). Press “Enter”; 12.Press “ENTER” , followed by “START”; 13.Stay with the centrifuge until full speed is attained. If you sense that the centrifuge is not running smoothly indicated by abnormal vibration, whining, or grinding noises, abort the run immediately and recheck the rotor lid and balance.
16 Centrifuge Standard Operating Procedure 7. TROUBLESHOOTING 7.1 Equipment Malfunction Users are not to make repairs. The centrifuge shall be maintained and repaired by qualified persons only. Table 7-1 lists some of the common minor problems that may occur and recommends the appropriate action for the user to take. Table 7-1: Centrifuge problems and causes.
17 Centrifuge Standard Operating Procedure Diagnostic Number/Message D13 – No rotor in chamber or drive problem Problem The is no rotor installed or the drive belt is loose or broken Result Run shuts down with maximum brake F1 and F2 – FRS, call service Required vacuum level not reached in allowed time Run shuts down with maximum break R1 and R2 – Rotor, ID problem No magnets identified, or magnets incorrectly identified Run continues, speed may be decreased.
18 Centrifuge Standard Operating Procedure Diagnostic Number/Message Problem Result R9 – Calibration error Rotor calibration error Run shuts down with maximum brake I1 – Rotor imbalance Rotor load is severely out of balance Run shuts down with maximum brake During lowtemperature runs (near -10oC) ice forms around the door opening Door will not open at the end of a run - C2, C3, C5, T1 through T4, D1 through D12, D14, D15, S1 through S14, H1 through H3 H5, H7, H8, H11 Call the Instrumentation
Centrifuge Standard Operating Procedure 19 8. PREVENTATIVE MAINTENANCE Users are not to perform maintenance. These procedures are carried out by the Instrumentation Technician. 8.1 Daily - Check the centrifuge interior for condensation Check the log book for any problems or concerns 8.
20 Centrifuge Standard Operating Procedure 9. QUICK REFERENCE GUIDE WEAR PERSONAL PROTECTIVE EQUIPMENT Eye and face protection, heat resistant gloves, and lab coat Step Button Action 1 POWER Turn the power switch on (|). 2 Foot Pedal 3 4 5 Install the rotor according to the manual – insure that the rotor is BALANCED and SECURELY in place. Then close the door. ROTOR RPM/RCF 6 TIME 7 TEMP 8 9 Press the foot pedal to unlock the chamber door; lift the door open.
Centrifuge Standard Operating Procedure 21 10.REFERENCES Beckman Coulter. 1999. JA 30.50 Ti Fixed Angle Rotor. Spinco Business Center of Beckman Coulter, Inc.; Palo Alto, California. Beckman Coulter. 2000. J-Lite JLA-10.500 Fixed Angle Rotor Assembly. JS-13.1 Swinging Bucket Rotor Manual. Beckman Coulter; Fullerton, California. Beckman Coulter. 2002. Avanti J-E Centrifuge Instruction Manual. Centrifuge Instrument Systems Development Center of Beckman Coulter, Inc.; Palo Alto, California. Beckman Coulter.
Centrifuge Standard Operating Procedure APPENDIX 1: ARABIDOPSIS CHLOROPLAST ISOLATION AND CHLOROPHYLL DETERMINATION This procedure is based on a method used by the M. Smith lab, Oct 2007. Required Buffers 1M HEPES-KOH -add 119.15g HEPES to ~300 mL of milli-Q H20 -stir until dissolved; add milli-Q to 500 mL -Adjust pH to 7.5 using 5M KOH -Autoclave @ L30 HEPES SORBITOL Buffer (HS Buffer, 200 mL) 10mL 1M HEPES-KOH, pH 7.5 (50mM final) 12.02g Sorbitol (330mM final) -adjust pH to 7.
Centrifuge Standard Operating Procedure 23 10 x Import Master Mix (50 mM HEPES, 330 mM Sorbitol, 400 mM KOAc, 50 mM MgOAc) For 25 mL: 0.298 g HEPES 1.503 g sorbitol 0.981 g KOAc 0.268 g MgOAc Adjust pH to 8.0 using KOH Stored as 1 mL aliquots @ -20°C Part I: Chloroplast Isolation 1. Prepare equipment; a. thaw Percoll b. cool down centrifuge (put in JLA 10.5 rotor and spin with at least two water blanks at 1,000 x g for 5 min; rotor stored in cold room) c.
Centrifuge Standard Operating Procedure 24 and then fill with HS), cover centrifuge tube with parafilm and gently invert 1x. 13.Centrifuge @ 1,000 x g, 6 min, 4C in swinging bucket rotor (JS13.1, MAX acceleration/deceleration). 14.Decant SN (can go down drain). 15.Re-suspend pellet in ~200-300 μl of HS buffer and transfer to 1.5 ml microcentrifuge tube, while estimating the total volume (using pipette). Part II: Chloroplast Concentration 1. Prepare reference sample: a. 10 μl HS buffer b.
Centrifuge Standard Operating Procedure APPENDIX 2: CENTRIFUGE USER LOG 25
26 Centrifuge Standard Operating Procedure DATE NAME EXT # SUPERVISOR ROTOR RUN TYPE DETAILS PROBLEMS / COMMENTS
Centrifuge Standard Operating Procedure APPENDIX 3: PREVENTATIVE MAINTENANCE LOG 27
28 Centrifuge Standard Operating Procedure DATE NAME EXT # TYPE OF MAINTENANCE FREQUENCY OF MAINTENANCE (I.E.
