P-9 7 Flaming/Brown™ Micropipette Pull er Operation Manual Rev. 2.46 - INT ( 20200630) One Digital Drive Novato, CA 94949 Voice: 415-883-0128 Web: www.sutter.com Fax: 415-883-0572 Email: info@sutter.com ii Copyright © 2020 Sutter Instrument Company. All Rights Reserved. Flaming/Brown™ is a trademark of Sutter Instrument Company. P-97 FLAMING/BROWN MICROPIPETTE PULLER OPERATION MANUAL – REV. 2.46 - INT (20200630) iii DISCLAIMER The pipette puller Model P-97 is designed for the specific use of creating micropipettes and no other use is recommended. This instrument creates items that should only be used in a laboratory environment for use on animal tissues. It is not intended for use, nor should be used, in human experimentation, or applied to humans in any way. This is not a medical device. Do not open or attempt to repair the instrument without expressed and explicit instructions from Sutter Instrument Company. Extreme heat and high voltages are present and could cause injury. Do not allow unauthorized and or untrained operatives to use this device. Any misuse will be the sole responsibility of the user/owner and Sutter Instruments assumes no implied or inferred liability for direct or consequential damages from this instrument if it is operated or used in any way other than for which it is designed. SAFETY WARNINGS AND PRECAUTIONS Electrical  Operate the P-97 using 110-120 V AC (60 Hz) or 220-240 V AC. (50 Hz) line voltage. This instrument is designed for connection to a standard laboratory power outlet (Overvoltage Category II), and because it is a microprocessor--controlled device, it should be accorded the same system wiring precautions as any 'computer type' system. A surge protector and power regulator are recommended.  Fuse Replacement: Replace only with the same type and rating: At 110 - 120 VAC: Type: Medium Time Delay or Time Lag Rating: 3A 250V (3 Amps, 250 Volts) Examples: Bussmann GMC-3A or GMC-3-R; or Littelfuse 239 003 or 239 003.P At 220 - 240 VAC: Type: Time Delay (IEC 60127-2, Sheet III) Rating: T2A_250V (2 Amps, 250 Volts) Examples: Bussmann GDC-2.0A, S506-2A, or S506-2R; or Littelfuse 218 002 or 218 002.P) A spare fuse is located in the power input module. Please refer to the fuse-replacement appendix for more details on fuse ratings and for instructions on how to change the fuse. Avoiding Electrical Shock and Fire-related Injury  Always use the grounded power supply cord set provided to connect the system to a grounded outlet (3-prong). This is required to protect you from injury in the event that an electrical hazard occurs. P-97 FLAMING/BROWN MICROPIPETTE PULLER OPERATION MANUAL – REV. 2.46 - INT (20200630) iv  Do not disassemble the system. Refer servicing to qualified personnel.  To prevent fire or shock hazard do not expose the unit to rain or moisture. Back Injury Prevention To avoid injuring your back or limbs it is recommended that you do not attempt to lift this instrument by yourself. The P-97 Micropipette Puller weighs in excess of 18 kg (over 39 lb) and should be moved by TWO (2) people. Operational Failure to comply with any of the following precautions may damage this device. • Operate the P-97 using the indicated line voltage. • The P-97 is designed for operation in a laboratory environment (Pollution Degree II) and at temperatures between 5°C - 40°C. • The P-97 is designed for connection to a standard laboratory power outlet (overvoltage Category II) with main supply voltage fluctuations not to exceed ± 10% of the normal voltage. • This unit is not designed for operation, nor has it been tested for safety, at altitudes above 2000 meters (6562 feet). • This unit was designed to operate at maximum relative humidity of 80% for temperatures up to 31°C decreasing linearly to 50% relative humidity at 40°C. • Operate only in a location where there is a free flow of fresh air on all sides. The fan draws air in through the vents on the sides and exhausts out both ends of the heat sink. NEVER ALLOW THE FREE FLOW OF AIR TO BE RESTRICTED. • To avoid burns do not touch the heating filament, the brass clamps holding the filament, or the heated ends of glass pipettes that have been pulled. • Only use Sutter Instrument Company replacement heating filaments. Handling Micropipettes Failure to comply with any of the following precautions may result in injury to the users of this device as well as those working in the general area near the device. • The micropipettes created using this instrument are very sharp and relatively fragile. Contact with the pulled micropipette tips, therefore, should be avoided to prevent accidentally impaling yourself. • Always dispose of micropipettes by placing them into a well-marked, spill-proof “sharps” container. • Use only with capillary glass (tubing) recommended by Sutter Instrument (see 1.2.1 Glass Capillary). P-97 FLAMING/BROWN MICROPIPETTE PULLER OPERATION MANUAL – REV. 2.46 - INT (20200630) v TABLE OF CONTENTS DISCLAIMER ...................................................................................................................................... iii SAFETY WARNINGS AND PRECAUTIONS ................................................................................... iii Electrical .................................................................................................................................................i ii Avoiding Electrical Shock and Fire-related Injury .............................................................................i ii Back Injury Prevention ......................................................................................................................... iv Operational .............................................................................................................................................i v Handling Micropipettes ......................................................................................................................... iv 1. GENERAL INFORMATION ........................................................................................................... 1 1.1 What’s New on the P-97? ................................................................................................................. 1 1.2 Glass Capillary & Heating Filament Specifications ...................................................................... 2 1.2.1 Glass Capillary ............................................................................................................................ 2 1.2.2 Heating Filament ....................................................................................................................... 2 1.3 Technical Support ............................................................................................................................. 2 1.4 System Description – Front Panel .................................................................................................. 2 1.4.1 Controls .......................................................................................................................................3 1.4.2 Display .........................................................................................................................................3 1.5 System Description – Mechanical (Puller Anatomy) .................................................................... 5 1.5.1 Some Basic Information ............................................................................................................ 5 1.5.2 Air Cooling System ..................................................................................................................... 5 1.5.3 Heating Assembly ...................................................................................................................... 5 1.5.4 Upper Pulley Assembly ............................................................................................................. 7 1.6 Cabinet ...............................................................................................................................................8 1.6.1 Electronics ...................................................................................................................................8 2. INSTALLATION .............................................................................................................................. 9 2.1 Unpacking ..........................................................................................................................................9 2.2 Setting Up ..........................................................................................................................................9 2.2.1 Line Power (Mains) .................................................................................................................... 9 3. OPERATING INSTRUCTIONS .................................................................................................... 11 3.1 First Time Use ................................................................................................................................1 1 3.2 Programs ..........................................................................................................................................1 2 3.2.1 Program Structure ...................................................................................................................1 2 3.2.2 Program Line Pull Cycle Parameters .................................................................................... 12 3.2.3 Pull Cycle ..................................................................................................................................1 4 3.2.4 Default Configuration ..............................................................................................................1 4 3.2.5 Selecting a Program (0 to 99) ................................................................................................. 14 3.2.6 Viewing a Program (< NEXT > / < LAST >) ..................................................................... 15 3.2.7 Clearing a Program from Memory (< CLR >) ..................................................................... 15 3.2.8 Editing a Program ....................................................................................................................1 5 3.2.8.1 Entering a New Program ................................................................................................. 16 3.2.8.2 Editing an Existing Program ........................................................................................... 16 3.3 Software Control Functions ...........................................................................................................1 6 3.3.1 RAMP TEST < 1 > .................................................................................................................1 7 3.3.2 CHANGE AIR PRESSURE < 2 > ........................................................................................ 18 P-97 FLAMING/BROWN MICROPIPETTE PULLER OPERATION MANUAL – REV. 2.46 - INT (20200630) vi 3.3.3 AIR MODE < 3 > ....................................................................................................................1 8 3.3.4 AIR TIME AT START OF PULL < 4 > ............................................................................... 19 3.3.5 AIR TIME AT END OF PULL < 5 > ................................................................................... 19 3.3.6 RESET TIME AND DATE < 6 > .......................................................................................... 19 3.3.7 WRITE-PROTECT THE PROGRAM < 7 > ....................................................................... 19 3.3.8 MEMORY TEST < 8 > ...........................................................................................................2 0 3.4 Pulling Pipettes ...............................................................................................................................2 0 3.4.1 Procedures ................................................................................................................................2 0 3.4.2 Notes on Program Operation ..................................................................................................2 1 3.5 Parameter Adjustment ...................................................................................................................2 1 3.5.1 General Information ................................................................................................................2 1 3.5.2 Micropipette/Microinjection Needle Fabrication .................................................................. 22 3.5.2.1 HEAT ..................................................................................................................................2 2 3.5.2.2 PULL Strength ..................................................................................................................2 2 3.5.2.3 VELOCITY (Trip Point) ...................................................................................................2 2 3.5.2.4 TIME Mode (Cooling) .......................................................................................................2 3 3.5.2.5 Delay Mode (Cooling) ........................................................................................................2 3 3.5.2.6 PRESSURE Adjustment ..................................................................................................2 4 3.5.2.7 Filament Width ..................................................................................................................2 4 3.5.3 Patch Pipette Fabrication .......................................................................................................2 4 3.5.3.1 HEAT ..................................................................................................................................2 4 3.5.3.2 PULL Strength ..................................................................................................................2 5 3.5.3.3 VELOCITY (Trip Point) ...................................................................................................2 5 3.5.3.4 Cooling ................................................................................................................................2 5 3.5.3.5 PRESSURE ........................................................................................................................2 5 3.5.4 Step-by-Step Patch Pipette Programming ............................................................................ 25 3.5.5 Technical Tips for Pulling Micropipettes .............................................................................. 27 3.5.5.1 Regulating the Time it takes to pull a Sharp Pipette .................................................... 27 3.5.5.1.1 Pipette Position ...........................................................................................................2 7 3.5.5.2 Regulating the Length and Tip Size of a Sharp Pipette ............................................... 27 3.5.5.2.1 Filament Width ...........................................................................................................2 7 3.5.5.2.2 Air Flow .......................................................................................................................2 7 3.5.5.2.3 DELAY Mode of Active Cooling ................................................................................ 27 3.5.6 Fire Polishing............................................................................................................................2 8 3.6 Heating Filaments ..........................................................................................................................2 9 3.6.1 General Information ................................................................................................................2 9 3.6.2 Filament Positioning and Aligning ........................................................................................ 29 3.6.3 Box Filament ............................................................................................................................3 0 3.6.3.1 Positioning..........................................................................................................................3 0 3.6.3.2 Geometry ............................................................................................................................3 1 3.6.4 Trough Filament ......................................................................................................................3 1 3.6.4.1 Positioning..........................................................................................................................3 2 3.6.4.2 Geometry ............................................................................................................................3 2 4. MAINTENANCE ............................................................................................................................ 33 4.1 Cleaning ...........................................................................................................................................3 3 4.2 Heating Filament Replacement ....................................................................................................3 3 4.2.1 Filament Replacement (Step by Step) ................................................................................... 33 4.2.2 Air jet Position ..........................................................................................................................3 3 P-97 FLAMING/BROWN MICROPIPETTE PULLER OPERATION MANUAL – REV. 2.46 - INT (20200630) vii 4.2.3 Positioning the Filament in Relation to the Glass Capillary .............................................. 33 4.3 Pulley Adjustment ..........................................................................................................................3 5 4.4 Regeneration of Drierite Granules ................................................................................................ 36 4.4.1 Removing the Canister ............................................................................................................3 6 4.4.2 Replacing the Granules ...........................................................................................................3 6 4.4.3 Reinstalling the Canister .........................................................................................................3 7 5. TROUBLESHOOTING .................................................................................................................. 39 5.1 Controlling Pipette Tip Shapes .....................................................................................................3 9 5.1.1 Problem: What type of glass should be used? ....................................................................... 39 5.1.2 Problem: The resistance of pulled pipettes is too low. How can a higher-resistance pipette be pulled? ...............................................................................................................................3 9 5.1.3 Problem: Okay, but how can tips be made even smaller. .................................................... 39 5.1.4 Problem: How can the size of a patch-pipette be increased? ............................................... 40 5.1.5 Problem: Patch-pipette tips vary in size from pull to pull. .................................................. 40 5.1.6 Problem: Difficulty making an injection pipette with a 0.5 – 1 µm tip that also has a very short final taper and tip (20 to 50 mm long). How can this be done? .......................................... 40 5.1.7 Problem: The electrodes are bent. How can they be made to pull straight? ..................... 40 5.1.8 Problem: One electrode is much longer than the other electrode. ..................................... 41 5.1.9 Problem: The shape and resistance of the pipette changes from pull to pull. ................... 41 5.2 Controller problems ........................................................................................................................4 1 5.2.1 Problem: The filament does not light up when pull is pressed. .......................................... 41 5.2.2 Problem: Display blank, fan not on, or locked up/overlap. .................................................. 42 5.2.3 Problem: Display shows only shows a cursor. ....................................................................... 42 5.2.4 Problem: Displayed program values are not correct ............................................................ 42 5.3 Technical Support ...........................................................................................................................4 2 APPENDIX A. Limited Warranty ...................................................................................................... 43 APPENDIX B. Accessories ................................................................................................................. 45 Filaments ...............................................................................................................................................4 5 Capillary Glass Tubing .........................................................................................................................4 5 Other Accessories ..................................................................................................................................4 5 APPENDIX C. Fuse Replacement ..................................................................................................... 47 APPENDIX D. Technical Specifications ............................................................................................ 49 APPENDIX E. Drierite Material Safety Data Sheet ......................................................................... 51 INDEX ................................................................................................................................................. 53 TABLE OF FIGURES Figure 1-1. P-97 Front panel. .................................................................................................................... 2 Figure 1-2. Program Display. .................................................................................................................... 3 Figure 1-3. P-97 base plate (top view, cover removed). .......................................................................... 5 Figure 1-4. Filament Block Assembly. ..................................................................................................... 6 Figure 1-5. Upper cable pulley assembly.................................................................................................. 7 Figure 2-1. P-97 Cabinet (rear view), showing power entry module. ................................................. 10 P-97 FLAMING/BROWN MICROPIPETTE PULLER OPERATION MANUAL – REV. 2.46 - INT (20200630) viii Figure 2-2. P-97 Cabinet (end view, left), showing power switch. ...................................................... 10 Figure 2-3. Power connection. ................................................................................................................. 10 Figure 3-1. Loading a piece of glass. ....................................................................................................... 11 Figure 3-2. Power-on display. .................................................................................................................. 14 Figure 3-3. P-97 Program display. .......................................................................................................... 15 Figure 3-4. Clear Program Display. ........................................................................................................ 15 Figure 3-5. Access to control functions. ................................................................................................. 16 Figure 3-6. Write protection default state. ............................................................................................. 20 Figure 3-7. Pull Cycle Report. ................................................................................................................. 20 Figure 3-8. Sample program. ................................................................................................................... 21 Figure 3-9. Pull cycle report (multiple-loops).. ...................................................................................... 21 Figure 3-10. Side view of fire polishing spacer. ..................................................................................... 28 Figure 3-11. Fire Polish Spacer in Puller Bar. ...................................................................................... 29 Figure 3-12. Filament alignment. ........................................................................................................... 29 Figure 3-13. End view of box filament and glass. .................................................................................. 30 Figure 3-14. Box filament positioning. ................................................................................................... 31 Figure 3-15. End view of trough filament and glass. ............................................................................ 31 Figure 3-16. Trough filament positioning. ............................................................................................. 32 Figure 4-1. V-Groove bearings and puller bars. .................................................................................... 33 Figure 4-2. Filament alignment. ............................................................................................................. 34 Figure 4-3. Micropipette shapes. ............................................................................................................. 35 Figure 4-4. Pulley Adjustment. ............................................................................................................... 36 Figure C-1. Power entry module. ............................................................................................................ 47 Figure C-2. Fuse holder. ........................................................................................................................... 48 TABLE OF TABLES Table 3-1. Standard factory configuration. ............................................................................................ 14 Table 3-2. Control Functions. .................................................................................................................. 17 Table 3-3. Maximum heat and recommended starting values for different filament shapes. ......... 18 Table 3-4. Box filament sizes. .................................................................................................................. 31 Table 3-5. Trough filament sizes. ............................................................................................................ 32 P-97 FLAMING/BROWN MICROPIPETTE PULLER OPERATION MANUAL – REV. 2.46 - INT (20200630)

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