SUTTER INSTRUMENT P-87 (01) PDF Document

To ensure safe operation and prevent damage or injury, please adhere to the following precautions:

On Operation:

• Operate the SUTTER INSTRUMENT P-87 using 110V a.c., 60Hz or 220V a.c., 50Hz-line voltage.

• The P-87 is designed to be operated in a laboratory environment (pollution degree I) and connect to a standard laboratory power outlet (overvoltage category II).

• This unit was not designed to be operated at altitudes above 2000 meters.

• Operate only in a location with free flow of fresh air on all sides. The fan draws air in through the side vents and exhausts out both ends of the heat sink. NEVER ALLOW THE FREE FLOW OF AIR TO BE RESTRICTED.

• As a microprocessor-controlled device, it should have the same system wiring precautions as any ‘computer type’ system, including line surge protection if required by other lab systems.

• Only use Sutter Instrument Company replacement heating filaments.

On Handling Micropipettes:

• The micropipettes created are very sharp and fragile. Avoid contact with the pulled micropipette tips to prevent accidental impaling.

• Always dispose of micropipettes by placing them into a well-marked, spill-proof “sharps” container.

• Use only with glassware recommended by Sutter Instrument Company.

General Safety Warnings:

• To prevent fire or shock hazard, do not expose the unit to rain or moisture.

• To avoid electrical shock, do not disassemble the unit. Refer servicing to qualified personnel. Always use the provided grounded power supply cord connected to a grounded outlet (3-prong).

• 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.

• The P-87 Micropipette Puller weighs in excess of 18kg. It is recommended that it be moved by TWO people to avoid injury.

The power cord connects to the Power Entry Module on the back of the unit, which also contains the Line Fuse and Voltage Selector Switch.

1. Confirm that the Voltage Selector Switch on the Power Entry Module is set to the proper value (110V a.c. or 220V a.c.). If not, turn the selector switch until the appropriate value aligns with the indicator. Note that the Line fuse differs for the two line voltages. If you change the voltage selector, you may also need to replace the fuse.

2. Ensure the Power Switch on the left end of the P-87 cabinet is turned OFF.

3. Plug the provided power cord into the Line Input socket on the Power Entry Module and then into a power source of the appropriate voltage and frequency.

1. Make certain that the SUTTER INSTRUMENT P-87 is plugged into a power outlet of the correct voltage and frequency.

2. Turn the Power switch on the left side of the cabinet ON.

3. Load a piece of the supplied sample glass into the puller as follows:

• Loosen the clamping knob.

• Place the glass in the V-groove in the puller bar, slide it beyond the clamp about 2 cm, and tighten the knob.

• Depress the spring stop on each puller bar to release them from their catch position.

• Pull both bars towards each other using the finger bars. Hold the bars in position so the hex head screw is touching the end of the slot in both puller bars.

• Loosen both clamping knobs, carefully slide the glass through the holes in the side of the heater chamber and into the V-groove of the opposite puller bar.

• Tighten down the clamping knobs.

4. Run a Ramp Test to determine a heat value that will melt your glass without burning out the filament. A Ramp Test should be run when using the puller for the first time, whenever you change the filament or glass, and before writing or editing a program.

To run the Ramp Test:

• Enter any program number <0-9>.

• Press clear <CLR> to enter the control functions.

• Press <0> to not clear all parameter values.

• Press <1> to run a RAMP TEST.

• Install glass and press <PULL>.

• Record the Ramp Test value shown on the display, as it will be used to set Program HEAT. To interrupt the RAMP TEST or reset the display, press <RESET>.

5. Enter <0> to display Program 0. The cursor will flash on the HEAT value. Enter the RAMP TEST value you just recorded for the HEAT parameter. Press the PULL key. The heating filament should turn on and the glass should separate in less than 10 seconds.

6. Loosen the clamping knobs and remove the pipettes from the puller bars.

LCD Display: Displays program parameters.

Reset: Re-initializes the controller.

Air Pressure: Sets the value of the air pressure during the active cooling phase of the pull cycle.

Keypad: Used to program parameter values and execute programs.

• 0-9: Used for choosing the desired program or control function, entering numeric values, and making YES/NO (1/0) decisions.

• CLR: Used to delete programs or numeric values. Also used to access the RAMP TEST.

• ENTR: Used to enter new values.

• NEXT: Used to move to the next line in a program while editing.

• LAST: Used to move to the previous line in a program while editing.

• PULL: Initiates the execution of a program.

• STOP: Aborts the execution of a program.

A program consists of one or more cycles, and each cycle has four programmable parameters:

HEAT (Range 0-999): Controls the level of electrical current supplied to the filament. The required HEAT is a function of the filament and glass type. It should be set relative to the Ramp Test value. Changes are typically made in steps of 5 units.

PULL (Range 0-255): Controls the force of the hard pull. Generally, a higher PULL value results in a smaller pipette tip diameter and a longer taper. Useful changes are 10 units or more.

VELOCITY (Range 0-255): This is the trip point for the hard pull. It measures the speed of the glass carriage system as the glass softens. It allows for the selection of a precise glass temperature to trigger the hard pull. Useful values range from 10 to 150. If VEL=0 and PULL=0, the puller enters a FIRE POLISH MODE where the HEAT is on for the duration specified by TIME.

TIME (Range 0-255): Controls the length of time the cooling air is active. If VEL>0, one unit of TIME represents 1/2 ms. If VEL=0, one unit of TIME represents 10 ms. If TIME=0, the air solenoid is disabled (no active cooling), which is useful for pulling special pipette shapes like long tubes for microinjection.

A typical PULL CYCLE in a PROGRAM follows these steps:

1. The heat turns on.

2. The glass heats up and a weak pull draws the glass out until it reaches the programmed velocity.

3. When the programmed velocity has been reached, the heat turns off and the air is turned on.

4. If TIME is >0, the hard pull (if any) is executed after a 40ms delay, and the air is activated for the specified TIME.

Selecting a Program

After powering on the instrument, the display will prompt “WHICH PROGRAM DO YOU WISH TO USE? (0-9)”. Press a number from 0 to 9 on the keypad to select one of the 10 storable programs. To select a different program later, press <RESET> to return to this prompt.

Viewing a Program

The display shows two adjacent lines of a program at a time. After selecting a program, the first two lines are shown. Use the <NEXT> key to view additional lines and the <LAST> key to scroll back to previous lines.

Clearing a Program from Memory

To clear values, press the <CLR> key. The display will ask, “DO YOU WISH TO CLEAR ALL VALUES FROM THE PRESENT CYCLE TO THE END? NO=0 YES=1”. Entering <1> will clear the program from the line the cursor is currently on to the end of the program. If the cursor is on Line 1, the entire program will be cleared.

Editing a Program (Entering a New Program or Editing an Existing One)

1. Select a program number. If it’s a new program, it’s best to clear it first.

2. The cursor will be blinking at the HEAT parameter on Line 1.

3. To enter a value (e.g., HEAT = 300), press the numbers <300>. The cursor will automatically move to the next parameter (PULL).

4. If you enter a value with fewer than three digits (e.g., VELOCITY = 90), you must press <ENTR> to enter the value and move the cursor to the next field.

5. To edit an existing value, use <NEXT> or <LAST> to scroll to the desired line. Press <ENTR> to tab the cursor to the field you want to change, and then enter the new value.

The software CONTROL FUNCTIONS are accessed through the <CLR> key.

1. Press <CLR>. The display will ask if you wish to clear values.

2. Press <0> (NO) to access the control functions menu.

3. The following functions are available:

RAMP TEST <1>

This test determines the HEAT value required to melt your specific glass with the installed filament. It should be run when using the puller for the first time, or whenever you change the glass or filament. When executed:

1. The puller increments the HEAT at 500 milliamps per second.

2. The puller bars move apart as the glass softens.

3. The HEAT turns off when a factory-set velocity is reached.

4. The display shows the RAMP TEST value, which is the HEAT value required to reach that velocity.

Press <RESET> to interrupt the test or reset the display afterwards.

Recommendations for HEAT settings based on Ramp Test:

• For Trough filaments: Recommended starting value = Ramp value + 5 units. Maximum program HEAT = Ramp value + 35 units.

• For Box filaments: Recommended starting value = Ramp value – 20 units. Maximum program HEAT = Ramp value + 30 units.

MEMORY TEST <2>

This performs a non-destructive test of the RAM. Press <RESET> to reset the system after the test.

1. Before pulling for the first time, run the RAMP TEST to find the appropriate HEAT value for your glass. Note the Ramp value reported.

2. Adjust the HEAT settings in your chosen program. A typical starting point is Ramp value plus 5 units.

3. Load the glass into the puller as described in the FIRST TIME USE section.

4. Press <0> on the keypad to view Program 0 (factory-installed micropipette program). Adjust the HEAT setting to your Ramp value plus 5 units.

5. Press the PULL key. The filament will heat, and the glass should separate within 10 seconds. The display will report the number of heating cycles (“loops”) and the last line used.

6. Loosen the clamping knobs and remove the newly formed pipettes.

The SUTTER INSTRUMENT P-87 can also loop through a program, which is useful for patch pipettes. Program 1 is a factory pre-programmed example of a multicycle program. When executed, the heating filament will cycle on and off repeatedly until the glass separates, forming a characteristic stubby geometry.

For fine micropipettes and microinjection needles, use a single-cycle program. Adjust the parameters as follows:

HEAT: Affects the length and tip size. Higher HEAT gives longer, finer tips. For trough filaments, start with Ramp value + 5. For box filaments, start with Ramp value – 20. Adjust the HEAT so the pull takes 5-6 seconds for the best micropipette reproducibility or 7+ seconds for microinjection needles.

PULL STRENGTH: Low values (40-75) give larger tips suitable for injection needles. High values (120-250) give smaller tips for micropipettes.

VELOCITY (trip point): Determines when the heat turns off. Typically set between 80-120 for micropipettes and 50-80 for microinjection needles.

TIME: Controls cooling air duration. The working range is narrow. For the finest tips, use a TIME value about 5 units higher than the lowest value that forms a tip. Adjusting air pressure is more effective for controlling tip length.

PRESSURE: Controls cooling air pressure. Higher pressure results in a shorter taper. Recommended values are ≤ 300 for thin-walled tubing and 500 for thick-walled tubing. Varying pressure around these values can control tip length.

Patch pipettes are formed using multiple heating cycles, often with a single-line looping program. Follow these steps to establish a program:

1. Run a Ramp Test with the glass you intend to use. Let the result be ‘R’.

2. Program one line of code as follows. Set PRESSURE to 500 for thick-walled glass and 300 for thin-walled glass.

*The VELOCITY value will need to be manipulated.

3. Insert your glass and execute the program. The program should “loop” multiple times (typically 3-5 loops). The display will report the number of loops.

4. Increase the VELOCITY in 5 to 10 unit increments and pull a pipette after each adjustment. As VELOCITY increases, the number of loops decreases.

5. Decrease the VELOCITY. As VELOCITY decreases, the number of loops increases.

6. Adjust VELOCITY to establish the desired number of loops to form your desired pipette shape. For reproducibility, set the final VELOCITY value midway between the values that result in one more and one less loop than desired.

7. For fine adjustments, convert your one-line, looping program into an equivalent multi-line program (e.g., a 4-loop program becomes a 4-line program with identical parameters on each line).

8. Now you can make adjustments to only the last or next-to-last line to fine-tune the pipette geometry:

• For larger diameter tips: Decrease HEAT in the last line.

• For smaller diameter tips: Increase or decrease VELOCITY in the next-to-last or last line by 5-10 units, OR add a small amount of PULL (10 or 20) to the last line.

The SUTTER INSTRUMENT P-87 can perform pseudo fire-polishing by using a program with a Velocity value of 0. The extent of polishing must be determined empirically.

1. Program the instrument for fire-polishing mode. A sample program is: Heat = Ramp value, Pull=0, Velocity=0, Time=250. In this mode, the Heat will turn on for the duration set by Time (1 Time unit = 10 msec). The above program will heat for 2.5 seconds.

2. Pull a pair of pipettes using your desired pulling program. After pulling, keep them clamped in the puller bars.

3. Reset the puller and select your polishing program (from step 1).

4. Manually push the puller bar (with the pipette) back towards the filament. Position the tip of the pipette just inside the edge of the filament. For consistent positioning, you can use the optional Fire Polishing Spacer, which fits into the slot in the puller bar and uses an adjustable screw to set the bar’s position.

5. Press Pull. The filament will heat for the programmed duration, polishing the end of the pipette. You may need to execute the program multiple times to achieve the desired polish.

6. When finished, remove the Spacer from the puller bar.

To maintain the SUTTER INSTRUMENT P-87 in optimal condition, use the vinyl dust cover whenever the unit is turned off.

• Occasionally clean the exterior and the base plate of the unit by wiping them with a dry cloth to remove dust and fine pieces of glass. Avoid contact with the filament.

• Occasionally, the V-groove pulleys and the edges of the pull bars which slide in their grooves must be cleaned to maintain reproducibility. This should be done using a dry cotton swab.

• DO NOT lubricate any components of the P-87.

1. Remove Old Filament: Loosen the two clamp screws that hold the old heater filament in place. Slide out the old filament.

2. Install New Filament: Slip in the new filament and center it over the air jet. Re-tighten the two clamp screws.

3. Check Air Nozzle Position: The air nozzle should be 2-3mm below the center of the filament. If not, loosen the screw holding the air nozzle and reposition it.

4. Position the Glass Tubing: The position of the glass relative to the filament is critical. Use the eccentric chrome screws on the aluminum angle plate to adjust the filament’s position. Loosen the flat head locking screws first.

• Turn the vertical chrome screw (A) to adjust the vertical position.

• Turn the horizontal chrome screw (B) to adjust the front-to-back position.

• Tighten the locking screws after adjustment.

5. Test the Left-to-Right Centering: After positioning, you must check if the filament is centered left-to-right over the air jet.

• Run a RAMP TEST with your glass and the new filament.

• With the pressure at 500, program a one-line program (e.g., HEAT=RAMP, PULL=120, VELOCITY=100, TIME=150).

• Pull a pair of pipettes. Remove them and hold them side by side. If the shanks of the pipettes vary in length, the filament is not centered. One pipette is receiving more cooling than the other.

• Loosen the filament clamping screws and move the filament very slightly towards the side that produced the shorter pipette. Tighten the clamps and pull again. Repeat this process until the pipettes are of equal length.

This adjustment should only be made if the two electrodes formed from one pull are of quite different lengths and you have already confirmed that the filament and air nozzle are correctly positioned. Unequal lengths can be caused by unequal cable tensions.

The goal is to equalize the tension on the two cables so that the puller bars rest against their stops without significant tension on the cables.

1. Hold the puller bars together with one hand.

2. Press on either cable between the carrier and the pulley. You should be able to feel about 2mm of deflection before the internal solenoid hits its stop.

3. If the deflection is more or less than 2mm, the pulley position should be changed.

4. Loosen the two screws above the pulley (J’) and turn the chrome eccentric screw (J) to move the pulley in small increments.

5. Adjust until the two cables have equal tension and the carriers come up against their stops (M) without significant tension. If there is too much tension, the initial pull will be inconsistent.

The Indicating Drierite (calcium sulfate) in the air-cooling system canister absorbs moisture, turning from blue to pink. When it is pink, it must be regenerated (dried).

1. Turn the puller off and unplug the power cord. Remove the puller cover.

2. Slide the input (left) and output (right) air tubes off the plastic connectors on the canister.

3. Release the two black plastic hold-downs that secure the canister and remove the canister.

4. Unscrew the aluminum end cap, being careful not to lose the black rubber sealing ring.

5. Remove the spring, its aluminum keeper, and the first filter to expose the Drierite.

6. Empty the exhausted granules from the canister. The far filter can then also be removed.

7. Spread the Drierite granules one layer deep on a tray and heat for one hour at about 200 degrees Celsius. Pre-dry the filters at 100°C for about 30 minutes.

8. Cool the granules in a tight container before refilling the plastic canister.

9. Reassemble the canister in reverse order: install the far filter, pour in the regenerated Drierite, insert the second filter and the keeper/spring assembly, and screw the cover back on, ensuring the rubber seal is flat.

10. Reinstall the canister on the baseplate. IMPORTANT: Install only the air input tube (larger tube, left-hand connector). Do not install the output tube yet.

11. Plug in the puller and turn it on. Let the air pump run for several minutes to blow air through the canister and into the room. This purges any Drierite dust that could clog the air solenoid or air jet.

12. After purging, turn the puller off, connect the output tube, and reinstall the puller cover.

If the controller fails to power up, check the line power fuse.

1. Unplug the power cord from the power entry module on the back of the controller.

2. This will reveal a slot just under the edge of the fuse holder. Use a screwdriver to pry the holder straight out.

3. The “active” fuse is readily visible. A spare fuse is stored in a concealed compartment within the holder.

4. To remove the spare fuse, press down on the end of the compartment and push it out of the other end. You can use the old fuse to push it out.

5. Replace the active fuse with the spare and re-install the fuse holder and power cord.

If the controller still fails to power up with the new fuse, contact technical support personnel.

The type of glass and its wall ratio (inside diameter to outside diameter) are two of the most important variables in controlling tip size.

In general, the thicker the wall in relation to the O.D. of the glass, the finer the tip will be. Conversely, the thinner the wall, the larger the tip will be. Thin wall glass generally gives the best results as it has the largest pore for a given tip size, leading to lower resistance and easier injection. However, for small cells, thin wall glass may not form tips fine enough for good penetration, in which case heavier wall glass must be used.

There is little correlation between tip size and electrode resistance; most resistance comes from the shank. A higher resistance electrode will likely have a longer shank and a smaller cone angle, which can aid in cell penetration.

1. Increase the HEAT value. This will generally decrease the tip size but also give a longer shank, increasing resistance. For 1.0mm O.D. x 0.5mm I.D. borosilicate glass, the finest tips are formed when the pull takes 5 to 7 seconds.

2. Increase the PULL strength. If the electrode is now too long with too high a resistance, increasing the pull strength can help. A pull strength of 125 will give tips less than 0.1µm. Increasing the pull to 250 will reduce tip size by about 5-10%. A pull of about 150 is recommended in most cases.

3. Adjust cooling. The last variable is the amount of cooling during the pull. If the pull takes more than 8 seconds, decreasing the cooling may somewhat decrease the tip size. Cooling is most effectively decreased by reducing air pressure, but a decreasing TIME value may also be useful.

1. The first thing to try is to reduce the HEAT. Try dropping the HEAT 5 units at a time to see if this will increase the size of the tips.

2. If that does not work, increase the air pressure in units of 50. The PULL should generally be set to 0 when pulling large-tipped (1-10 µm) pipettes.

3. Review the “Step-by-step patch programming” procedure in the Parameter Adjustment section for more detailed guidance.

This can happen when a pipette is formed in two or more loops (heating cycles). If the pipette is formed in three loops in one case and four loops on the next pull, the tips will not be the same.

This indicates that your VELOCITY setting is on the border between two loop counts. Adding or subtracting one unit from the VELOCITY value will likely cause the pipette to consistently form in the lower or higher loop count, respectively.

The best procedure for a reliable program is to find the VELOCITY value where the number of loops changes, and then pick a final VELOCITY value halfway between the values that produce the different loop counts. This will place your program in a stable region.

This problem occurs most often when using the trough filament. Switching to a box-type filament will produce straighter pipettes. The bend in the pipette has no effect on the tip itself, but if you are penetrating deep into tissue, the bend may lead the pipette to the wrong area. Note that a box filament is less effective for air cooling, so you give up some of the length control that the cooling air provides with a trough filament.

This is caused by one of two things:

1. The filament is not centered over the air jet. If it is off-center, one pipette receives more cooling than the other, resulting in different lengths. Follow the procedure “Testing the position” in the Heating filament replacement section of the Maintenance chapter to correct this.

2. The tension in the two pull cables is not equal. If the filament is correctly centered but the pipettes are still unequal, the cable tension needs adjustment. Follow the “Pulley Adjustment” procedure in the Maintenance chapter to equalize the tension.

1. Incorrect cable adjustment: In most cases, this is due to one or both of the puller cables being set too tight. If a cable is adjusted so that the carrier cannot come against its mechanical stop, the initial pull tension will depend on how hard the carriers are squeezed together when the glass is clamped. This leads to inconsistency. To fix this, see the pulley adjustment section in the Maintenance chapter.

2. Dirt on carrier bars or bearings: Clean the carriers and bearings with a lint-free tissue or cloth.

3. Glass variability: If the problem persists, run the ramp test several times using one long piece of glass, moving the glass over after each test. If the ramp values are +/- 4 units or less, the problem may be with the glass itself. If the values are greater than +/- 4 units, contact Sutter Instruments.

There are several possible reasons for this issue:

1. Check if the filament has burned out. A very fine break may be hard to see, so you may need to loosen the screws holding it to inspect it closely.

2. If the filament is okay, try running the ramp test. If you have just changed the filament, it may require a very different HEAT value than the previous one. Always run a ramp test after changing the filament.

3. If the ramp test reaches a HEAT value of 999 without the filament heating up, check the tightness of all exposed electrical connectors in the filament current pathway. This includes the filament holding screws, the screws holding the two brass jaws, and the two nuts connecting the filament wires to the posts at the back of the filament block.

4. If these connectors are tight, the problem may be with the power FETs on the heat sink. Contact Sutter Instruments for further instructions.

1. Check the power cord and the wall a.c. outlet.

2. If the unit is properly plugged in and still does not work, remove the power cord and check the fuse. If the fuse has blown, a failure in non-serviceable components has likely occurred. Contact Sutter Instrument Company Technical Support.

3. If the fuse is still good but the unit does not work, a failure in non-serviceable components has likely occurred. Contact Sutter Instrument Company Technical Support.

This problem can occur when the power has been turned off and on again too rapidly, causing the microprocessor to fail to initialize the display properly.

Press RESET. The display should show the proper power-up message. Always allow at least 5 seconds before turning the power back on.

If the display still shows a row of blocks after pressing RESET and waiting, a failure in non-serviceable components has likely occurred. Contact Sutter Instrument Company Technical Support.

1. Make sure that the values were not changed by another user. It is recommended to always write down your program values and the ramp-test value and keep them in a secure place.

2. If the values you entered are not held when the power is turned off, a failure in non-serviceable components has likely occurred. Contact Sutter Instrument Company Technical Support.