User`s Manual
Contents
1. Reliable microinjection also requires skill in making and using micropipettes Authorities on microinjection state that the most important single factor in microinjection is the micropipette The MicroJect 1000A Pico Injector with its reproducibility simplifies the search for the optimum micropipette toll free 800 272 2775 phone 508 893 8999 web www btxonline com BTX Harvard Apparatus MicroJect 1000A Pico Injector User s Manual Preliminaries Microinjection Microinjection also involves other skills several other instruments and accessories and various supplies The purpose of this section is to give an overview of these techniques for those new to microinjection some guidance on equipment selection is also supplied Required Auxiliary Equipment for Microinjection The required equipment includes a pipette puller and micromanipulator s Ideally the puller should be capable of making pipettes with tip diameters in the 0 2 to 1 5 micron range with a short enough taper length for both mechanical strength and low flow resist ance If most injections are to be extracellular then a puller suitable for extracellular patch clamp pipettes is satisfactory For intracellular injections some magnetic pullers may be suitable Alternatively a two stage gravity puller with variable weights can be satisfactory over the entire range The required three dimensional movement can be produced by an inexpensive mechanically linked micromanipulat2. again enter the cell membrane at right angles to avoid shearing Non spherical cells for example cultured fibroblasts have a more robust cytoskeletal structure so the pipette can be pushed in even if not perpendicular to the membrane surface For less firmly attached cells the injection pipette can be bent near the tip after pulling The pipette s main axis slopes slightly down from the horizontal The angle of bend should allow the tip to point straight down With an inverted microscope the tip is viewed through the cell as it is lowered for injection The microscope is focused on the cell s top surface and the tip comes into focus just before insertion Again shearing forces are avoided Suitable bends can be made with a microforge a simple way to do this is to move the pipette near a hot filament at the position of desired bend The tip will spontaneously bend away In all of the above techniques a three dimensional micromanipulator controls the movement of the injection pipette If this straight pipette is instead attached to a condenser mount inverted scope then a one dimensional manipulator can be used The remaining two directions of manipulation are done with stage microme ters moving the vertical injection pipette over each cell in turn If the vibrations transmitted with the condenser mounting are manageable then this approach gives the fastest rate of cell injection toll free 800 272 2775 phone 508 893 8999 web www
3. authorization number has been issued by our customer service department The customer is responsible for shipping charges Please allow a reasonable period of time for completion of repairs replacement and return If the unit is replaced the replacement unit is covered only for the remainder of the original warranty period dating from the purchase of the original device This warranty gives you specific rights and you may also have other rights which vary from state to state Repair Facilities and Parts BTX Harvard Apparatus stocks replacement and repair parts When ordering please describe parts as completely as possible preferably using our part numbers If practi cal enclose a sample or drawing CAUTION This apparatus is not registered with the FDA and is not for clinical use on human patients CAUTION Not for clinical use on human patients toll free 800 272 2775 phone 508 893 8999 web www btxonline com BTX Harvard Apparatus MicroJect 1000A Pico Injector User s Manual General Safety Summary Please read the following safety precautions to ensure proper use of your pico injector To avoid potential hazards and product damage use this product only as instructed in this manual If the equipment is used in a manner not specified by the manufacturer the protection provided by the equipment may be impaired To Prevent Hazard or Injury USE PROPER POWER SUPPLY The product is supplied with an approved power s
4. exempt from the RoHS Directive We will continue to monitor the application of the RoHS Directive to its products and will comply with any changes as they apply Do Not Dispose Product with Municipal Waste e Special Collection Disposal Required BTX Harvard Apparatus MicroJect 1000A Pico Injector User s Manual Table of Contents SUBJECT PAGE NO Warranty and Repair Information rrssnvvrnrnnnvennnnvvennnnvenennn 2 General Safety Summary rrrnnvrrnnnvvrnrnnnvnnnnnvvennnnvvnnsnnvesnnneer 3 SPEGINGAHNONS Lunnan 4 Ip Lue 101 eg 1198 EE SE anen auna aeaeaie 5 Preliminatles i cci2i cs cctcecesiectenecetens tel cceeenee i iescdceesedensenctieessastle 6 7 Front Panel Controls amp Connections rrnnrnnnnrnrnvnnnnvnnnn 8 9 Rear Panel Connectors amp Attachments rnnnnrnnnnnnnnrnnn 10 Using the MicroJect 1000A Pico Injector 0008 11 13 Micro Injection Substances Cell Types cceeeeee 14 Setting Pressure amp Time Pulse rrrnnrnnnnnvvrnnnnvvrnnnnnvnnnnvvnnnnn 14 Gas Usage Warning cccsccccscetiepesdece eae cscecensupe cde enespuues 14 HOSE CONMOCHIONS wc sisicccccecs satsstecesasctsccenncatseacedecectoceenereecdecs 14 Volume Calibration Chart errnvrnnnnvnrnvvnnevnnnnnvnnnnnnnnnnnnnr 15 Power Entry Module i ciitscics sisi csssticeiisicteciacienivins 16 toll free 800 272 2775 phone 508 893 8999 web www btxonline com BTX Harvard Apparatus MicroJect 1000A Pic
5. is not wanted turn off the switch on the rear panel Two output hoses are supplied with the MicroJect 1000A Pico Injector These are designed for any of the three optional pipette holders described in the PRICE LIST Connect the hoses to the chosen holder s as follows MicroJect 1000A Pico Injector Unscrew Hex Nut the end of the holder Output Hose with a 2 mm diameter Holder hole This end hex piece should be placed over the tube end Thread the hex piece on the holder and tighten Aeg ine lt lt Silicon Rubber Gasket 5mm L x 1mm ID 1mm OD capillary is inserted in the opposite lt lt re end of the holder ee through its o ring sili con rubber gasket and its fitting is then tightened Glass Tube Fig 3 MicroJect 1000A 1 0 mm OD Glass Pipette Assembly toll free 800 272 2775 phone 508 893 8999 web www btxonline com BTX Harvard Apparatus MicroJect 1000A Pico Injector User s Manual 12 Using the MicroJect 1000A Pico Injector MicroJect 1000A ENE Pico Injector Unscrew ule tks the end of the holder Haa SON with the 2 mm diameter OS 3 hole and place it over the output hose PLI OH Reattach this fitting securely with the hose inserted as far as it will 9o The 1 5 mm OD capillary is similarly attached to the other end of the holder through its o ring Ve Bushing Metal Sleeve Silicon Rubber Gasket 7 5mm L x 1 5mm ID Knurled Nut Glass Tube Fig 4 MicroJect 1000
6. 1 5 mm OD Glass Pipette Assembly Attach output and holding hose to the appropriate front panel gas ports Tighten them securely so that the valves within these ports are open allowing pressure to be controlled in the hose and connecting micropipette Micropipette Filling Filling can be done from the tip of the micropipette or with a few extra steps from the barrel of the pipette Filling from the tip is most suitable if a filling fiber is deliber ately not being used due to irregularities in the balancing see BALANCE below Simply prepare a bolus of injection material on a glass slide and maneuver the tip into it using a micromanipulator Depress the FILL button to apply about 12 psi 82 kP of suction continuously as long as needed If evaporation of a small bolus is a concern it can be prepared under oil and the micropipette tip similarly moved into it Filling can be done from the back end barrel of the pipette using a syringe with a thin hypodermic needle inserted so its tip is down in the tapered section If the capil lary has a filling fiber attached to the inner wall the pipette tip will then fill by capillary action without air bubbles at least for larger tips Variations on this procedure involve filling a smaller capillary first and inserting it for the back fill Balance The inflow into the pipette caused by capillary forces prior to or in between injections should be a small percentage of that being injected As the vol
7. MicroJect 1000A Pico Injector User s Manual Item No System 45 0758 MicroJect 1000A Basic System 45 0759 MicroJect 1000A Plus System 45 0760 MicroJect 1000A Max System BTX HARVARD APPARATUS The Electroporation Experts Publication 5506 001 REV A WEEE RoHS Compliance Statement EU Directives WEEE and RoHS To Our Valued Customers We are committed to being a good corporate citizen As part of that commitment we strive to maintain an environmentally conscious manufacturing operation The European Union EU has enacted two Directives the first on product recycling Waste Electrical and Electronic Equipment WEEE and the second limiting the use of certain substances Restriction on the use of Hazardous Substances RoHS Over time these Directives will be implemented in the national laws of each EU Member State Once the final national regulations have been put into place recycling will be offered for our products which are within the scope of the WEEE Directive Products falling under the scope of the WEEE Directive available for sale after August 13 2005 will be identified with a wheelie bin symbol Two Categories of products covered by the WEEE Directive are currently exempt from the RoHS Directive Category 8 medical devices with the exception of implanted or infected products and Category 9 monitoring and control instruments Most of our products fall into either Category 8 or 9 and are currently
8. SSURE METER SELECT switch A negative sign indicates suction 7 PRESSURE UNITS toggle switch Toggle switch controls injection pressure display unit of measure Injection pressure can be displayed in either pound force per square inch PSI or in kilopascals kP 1 psi 6 89 kP 8 RESET pushbutton This button resets the injection count display to zero 9 INJECTION COUNT display This four digit display 0 9999 gives the total number of injections triggered manually by the panel pushbutton or footswitch or electrically since the last reset toll free 800 272 2775 phone 508 893 8999 web www btxonline com BTX Harvard Apparatus MicroJect 1000A Pico Injector User s Manual Front Panel Connectors amp Controls 10 PRESSURE METER SELECT switch This switch selects various places inside the PICO INJECTOR for reading the pressure It does not change the pressure applied to the output PINJ is the pressure applied to pressure applied at the output during injection PBAL is the pressure applied to the pipette when injection is not taking place PCLR is the pressure externally supplied to the input on the rear panel and used the CLEAR mode PFILL is the negative pressure applied to fill the injection pipette from the tip POUT is the pressure currently applied to the output port 11 P INJ regulator This seven turn control is used to set the injection pressure over the range from about 0 4 to 60 0 psi about 2 8 413 kP Clockwise rot
9. ation increases the pressure Locking nut should be loose to avoid damaging the shaft s threads if changing pressure constantly 12 POWER rocker switch Controls power to the unit 13 TRIG IN connector This BNC connector is for electrically initiating injection A positive TTL level pulse is required baseline 0 0 V trigger 5 V lasting at least 50 microseconds 14 GATE IN connector This BNC connector is for external timing of the duration of injection A positive TTL level pulse see 13 is required with the duration of injection determined by the duration of the applied pulse 15 TRIG GATE OUT connector This TTL output see 13 can be used to trigger or synchronize other electrical instruments Its duration is that of the injection 16 PRESSURE MONITOR out This BNC connector delivers an electrical level whose amplitude gives the actual pressure applied to the meter The conversion factor is 10 mV psi or 1mV KPa 17 FILL VACUUM regulator This regulator sets the vacuum applied to the POUT connector The range is 0 to 12 PSI 0 to 82KPa regulated 18 POUT connector This connector is attached to the injection pipette using the supplied output hose PLI OH Without the hose attached the port is closed In use the injection balance fill and clear pressures are delivered here 19 PHOLD regulator This regulator sets the low suction pressure applied to the PHOLD connector Counterclockwise increases the pressure Like the ot
10. btxonline com BTX Harvard Apparatus MicroJect 1000A Pico Injector User s Manual Front Panel Connectors amp Controls Figure 1 Front Panel Refer to Figure 1 for the location of controls and connectors Numbers refer to the order of listing below 1 INJECT pushbutton Push this to manually trigger the injection pressure for a time set by the internal timer The switch remains lit for the duration of the injection 2 INJECT TIME SETTING DIAL This dial controls the injection time setting Turning the dial changes the injection time in seconds pressing the dial in and turning changes the injection time in fractions of a second 2 1 INJECT TIME Display Displays injection time in seconds 3 CLEAR pushbutton Push this briefly to deliver a half second pressure surge to clear a clogged pipette The pressure applied is the supply pressure If the button is left pushed in for longer than a half second the clearing surge is extended The button remains lit for the duration of the clear 4 FILL pushbutton Push the button to apply suction to the delivery pipette to fill it from the tip The suction is applied as long as this button is held and LED is lit 5 HOLD pushbutton Push this button to apply sufficiently small suction to holding pipette LED is lit to hold a cell for injection Push this a second time to drop this suction to zero 6 PRESSURE display This three digit display gives the gauge pressure selected by the PRE
11. en liquid is then raised to cover the tip The inflow volume is approxi mately the difference between the volume of two cones The estimate is more precise for smaller initial volumes in the pipette Clear To clear a clogged micropipette push the CLEAR button momentarily once the tip has been removed from a cell Watch the tip region while this is done to see the brief movement of liquid denoting that the tip is clear Repeat if needed The clearing pressure pulse has a preset duration of 500 msec to avoid excessive release if the clog clears immediately Repeat as needed Hold To hold a suspended cell for injection attach a suitable holding pipette to the pipette holder holding hose Momentarily attach to the P ur port Backfill the holding pipette using the fill function switch with fluid so that tube length is half to three quarters filled Disconnect the holding hose connector from the P pyr port and attach to the P hop Port Set the hold pressure control to minimum and move the micropipette to the cell using a micromanipulator Turn on the HOLD panel switch and increase the hold pressure as needed When finished with that cell turn off the hold pressure with the panel or footswitch If more cells of the same kind are being injected then subse quent holds require only the ON OFF control of the switch If the cell doesn t drop off the holding pipette tip when hold is turned off a brief gentle squeeze of the rubber bulb located on t
12. he holding output hose should release it Note Be careful not to overfill the hose to avoid liquid going into the pneumatic system inside the unit Footswitch es Optional footswitches can be used for inject fill and turning on and off the hold function These switches are plugged into the rear panel each is wired in parallel with the corresponding front panel switch See the instructions for each panel switch to see how they operate When a footswitch is used with the GATE connector the duration of injection is manually determined by the duration of pressing the footswitch In some applications users may prefer to set up the duration of injection manually connect the MONITOR output to an oscilloscope or fast enough strip chart recorder to measure this time and then switch to the internal timer for subsequent injections Electrical Inputs Outputs 13 16 in Figure 1 These connectors allow additional control especially when synchronizing injection to other stimulations or recording See items 13 16 on pages 8 and 9 for details In partic ular the PRESSURE MONITOR output signal is a precise measure of relative injection volume for a given pipette Since the volume delivered is linear in the net injection pressure P w P ga and injection time the area under a graph of this signal versus time is a measure of the relative volume Injection Volume Adjustment Adjust injection volume by changing either injection pressure injection dura
13. her pressure controls the current rotational position is uniquely related to the pressure The range is 0 0 5 kP 5 cm height of water 20 PHOLD connector The supplied holding hose is attached to this connnector The suction set by the PHOLD regulator is applied here to hold a suspended cell for injection 21 PBAL regulator This regulator sets the balance pressure over the range from 0 1 to 10 psi about 0 7 70 kP Clockwise rotation increases the pressure 22 AUDIO MUTE switch Controls injection sound toll free 800 272 2775 phone 508 893 8999 web www btxonline com BTX Harvard Apparatus MicroJect 1000A Pico Injector User s Manual 10 Rear Panel Connectors amp Attachments gt MICROJECT 1000A eg aan _ im Ansi ees gt ce p 1 Figure 2 Rear Panel 1 Foot Switch Inputs Connectors An optional footswitch PLI FS can be connected to any one of these connectors When connected to HOLD pushing the footswitch once turns on the suction for holding a cell Pushing it a second time turns that suction off When connected to INJ pushing the footswitch starts injection When connected to FILL the filling suction is applied as long as the footswitch is depressed When connected to GATING injection pressure continues as long as the footswitch is depressed 2 Pressure Input Connector To have all the functions working the instrument needs 70 to 105 psi To have only injection 70 ps
14. i is needed This connector is the input for the compressed gas See PRELIMINARIES for recommendations on the gas A maximum of 105 psi can be safely applied 100 psi is optimal At lower pressure gas will be used at a slower rate Avoid input pressures less than 100 psi if the FILL function is being used 3 Input Filter The PICO INJECTOR is supplied with an input filter It traps particles larger than 0 5 micron as well as the liquid often present in a building s compressed air lines Drain by pushing the button on the underside of its transparent case Through a hole located in the bottom case 4 Filter Window Allows for easy viewing of filter to determine when it needs to be drained 5 Power Input Connector Jack for universal AC input 9 volt 6 watt DC output power supply toll free 800 272 2775 phone 508 893 8999 web www btxonline com BTX Harvard Apparatus MicroJect 1000A Pico Injector User s Manual 11 Using the MicroJect 1000A Pico Injector General Considerations It is much easier to reliably inject large volumes than small ones For large volumes the balance pressure capability is not needed Pipettes seldom clog so the clear capa bility is also not needed The dividing line between large and small is not rigid it depends on how quantitative a delivery is required That volume line would typically be in the 10 100 pLiter range For convenient visualization and approximate geometric measurement 1 fLiter
15. ipette BALANCE A lower balance pressure applied to the delivery pipette between injections prevents clogging caused by pipette movement as well as dilution of the injected material by capillary action CLEAR Momentary application of high pressure can be used to clear clogged pipettes Triggering of injection is accomplished three ways panel push button optional foot switch or an externally applied electrical signal TTL pulse The duration of injection can be determined by an internal clock set with a digital switch or can be gated by the duration either of an externally applied electrical pulse or of the depression of an optional foot switch This manual assumes the reader has no previous experience with microinjection either extracellular or intracellular Those planning on larger volume delivery 100 pL and up can ignore the BALANCE feature Some reference will be made to a less precise technique for smaller volume delivery to highlight the precision determining features of this instrument This technique involves pressurizing the gas ina macro syringe attached to a delivery micropipette by advancing the piston of the syringe with a micrometer screw Ejection takes place continuously from the pipette the volume delivered depends both on the unregulated over pressure in the syringe and the poorly known time the pipette is left inside the cell This continuous ejection technique supposedly reduces the frequency of pipette clogging
16. is a cube 1 micron ona side or a sphere 1 24 microns in diameter 1 pLiter is a cube 10 microns on a side or a sphere 12 4 microns in diameter while 1 nLiter is a cube 100 microns on a side or a sphere 124 microns in diameter Because volume goes as the cube of linear dimensions such geometric volumes are imprecise but often useful Extracellular delivery is nearly always large Intracellular is often of small volumes but not for frog oocytes A more quantitative way to distinguish between large and small is given in the balance section below Interconnections and Initial Set Up Connect the gas input hose to the rear panel input connector of the MicroJect 1000A Pico Injector Connect the other end with optional input hose adapter if needed to the gas supply Turn on the POWER switch and verify the input gas pressure with the digital meter by setting the PRESSURE METER SOURCE switch to PCLR Practice with the inject and balance pressure controls by first turning the METER SELECT switch and then adjusting each in turn With non zero values of inject and balance pressure set the PRESSURE METER SOURCE switch to POUT NOTE The PRESSURE METER SELECT switch must be set to POUT to read changes in pressure on the display or MONITOR output Set the inject time to five seconds and push the panel INJECT switch to see the temporary change in pressure from balance to inject and back A buzzer will sound during injection If this
17. iters 224 40 Nanoliters ca Pressure Time Pipette Tip Q p s i sec 1 D pm Femtoliters Picoliter Nanoliter Microliter Milliliter 1 1 0 1 179 52 0 18 2 10 1 0 1 1795 20 1 80 T 20 1 0 1 3590 40 3 59 30 1 0 1 5385 60 5 39 O 40 1 0 1 7180 80 7 18 3 2 50 1 0 1 8976 00 8 98 lt 60 1 0 1 10771 20 10 77 2 1 1 1 17952 0 18 gt 10 1 1 1795 20 1 80 5 20 1 1 3590 40 3 59 Q 30 1 1 5385 60 5 39 O 40 1 1 7180 80 7 18 50 1 1 8976 00 8 98 60 1 1 10771 20 10 77 5 1 1 5 2244 002 O 10 1 5 224 40 022 gt 20 fi 448 80 0 45 g 30 1 5 673 20 0 67 5 40 1 5 897 60 0 90 5 50 1 5 1012200112 60 1 5 1346 40 1 35 T 1 1 10 179 52 0 18 D 10 1 10 1795 20 1 80 20 1 10 3590 20 3 59 30 1 10 5385 60 5 39 40 1 10 7180 80 7 18 50 1 10 8976 00 8 98 60 1 10 10771 20 10 77 1 1 75 75 74 0 08 10 1 75 757 35 0 76 20 1 75 1514 70 1 51 30 1 75 2272 05 227 40 1 75 3029 40 3 03 50 1 75 3786 75 3 79 60 1 75 4544 10 4 54 Constant includes unit conversion factors and effects of water like viscosity and needle taper for a cone half angle of 10 medium taper 15 toll free 800 272 2775 phone 508 893 8999 web www btxonline com BTX Harvard Apparatus MicroJect 1000A Pico Injector User s Manual 16 Power E
18. lear injections a smaller taper angle is needed to avoid leakage further up the shank of the pipette at the plasma membrane Although even intracellular injection can be done from below with an upright microscope most injections are done from the side or from above the cells Four different strategies have been used to suitably fix the cell in position for successful intracellular injection For suspended cells a second larger pipette is used to hold the cell This pipette s tip is first polished with a microforge done by placing the pipette within 5 microns of a hot filament for a few seconds With its axis horizontal it is moved to hold the cell with applied suction The injection pipette is also straight and is inserted hori zontally from the opposite side This geometry avoids damage to the cell mem brane caused by shearing forces The optics are straightforward because the pipettes remain in focus as they are advanced For cells that can be or are attached to a surface in a closely packed layer straight injection pipettes can also be used In this case the pipette axis slopes slightly down from the horizontal The tendency of the cells to slide when the pipette enters is resisted by the extracellular environment or attachment to the culture sur face The microscope should be focused on the cell s surface The pipette tip then comes into focus just before injection If the cell is nearly spherical the hardest case the pipette should
19. ntry Module PEM The Microject 1000A is equipped with a universal AC input 9 volt 6 watt DC output power supply with interchangeable input blades Figure 1 Wall adapter with interchangeable input blades Figure 3 Install input blade 1 Slide the blade lock in the direction of the arrow with one hand and lift out the contact protector with your other hand Align the rounded corners of the input blade being installed with the rounded corners on the adapter Lower the square corners of the blade into the adapter Pull back on the blade lock and lower the rounded corners into adapter release the blade lock Figure 4 Complete assembly toll free 800 272 2775 phone 508 893 8999 web www btxonline com There are many reasons to visit our website online at any time Register for newsletters and technical updates View a complete listing of BTH distributors worldwide Contact us for a quotation or technical support Search our products Submit protocols To Order contact BTH customer service today toll free 800 272 2775 usa amp Canada phone 508 893 8999 fax 508 429 5732 customer technical support e mail and website HARVARD apparatus techsu pport btx harvardapparatus com The Electroporation Experts WWW b t X 0 n in e a C 0 m
20. o Injector User s Manual Warranty amp Repair Information Serial Numbers All inquires concerning our product should refer to the serial number of the unit Serial numbers are located on the rear of the chassis Calibrations All electrical apparatus is calibrated at rated voltage and frequency Warranty BTX Harvard Apparatus warranties this instrument for a period of one year from date of purchase At its option BTX Harvard Apparatus will repair or replace the unit if it is found to be defective as to workmanship or material This warranty does not extend to damage resulting from misuse neglect or abuse normal wear and tear or accident This warranty extends only to the original customer purchaser IN NO EVENT SHALL BTX HARVARD APPARATUS BE LIABLE FOR INCIDENTAL OR CONSEQUENTIAL DAMAGES Some states do not allow exclusion or limitation of incidental or consequential damages so the above limitation or exclusion may not apply to you THERE ARE NO IMPLIED WARRANTIES OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR USE OR OF ANY OTHER NATURE Some states do not allow this limitation on an implied warranty so the above limitation may not apply to you If a defect arises within the two year warranty period promptly contact BTX Harvard Apparatus Inc 84 October Hill Road Building 7 Holliston Massachusetts 01746 1371 using our toll free number 1 800 272 2775 Goods will not be accepted for return unless an RMA returned materials
21. or for large cells such as frog oocytes More commonly a hydraulic one for fine vibration free movement is mounted on a coarse mechanical manipulator Suitable equipment is available from Harvard Apparatus Inc Required Supplies These are compressed gas and microcapillaries Compressed air is suitable for oxygen insensitive injection material Nitrogen is a satisfactory inert gas for the general case A pressure of 105 psi is sufficient a regulator will be needed if supplied from a bottle of compressed gas Optional Equipment for Microinjection This includes a microforge to bend a micropipette or to polish a pipette tip for holding a cell a microgrinder to bevel the pipette tip to increase the delivery rate without addi tional cell damage and an micro incubator to hold the cells at incubation temperatures during microinjection Suitable equipment is available from Harvard Apparatus Inc Accessories These are also available to enhance your MicroJect 1000A Pico Injector see Price List Micropipettes The micropipettes are made with a pipette puller from a microcapillary 1 2 mm in diameter by heating some 3 10 mm of its length with a concentric heater while applying a force gravitational or magnetic to pull both ends of the capillary apart Two micropipettes are produced per capillary 1 Distinguishing Parameters Two useful distinguishing parameters of the micropipette are the inside diameter of its tip and the angle of
22. pillary tube with a solution medium This serves as a buffer when the holding function is 2Z preventing a constant vacuum at the pipette tip caused by capillary action Gas Usage Warning To provide finely controllable output pressure the gas regulators are of the bleeding type Such regulators use gas even in the absence of ejections The MicroJect 1000 Pico InjectorA thus uses gas even when off To eliminate this consumption and as a good safety practice turn off the gas supply at the source when the MicroJect 1000A Pico Injector is not in use The suction functions HOLD and FILL use much more gas than INJECT and BALANCE Plan accordingly Notice that the BALANCE is on continuously once an output hose is attached to the front panel without a micropipette attached even this gas usage can be significant Hose Connections The input output and holding hoses should be attached to their respective connectors If each connector s needle valve located in the micro injector body is not fully opened the airflow will be restricted or blocked To prevent this from happening check each connector for tightness by turning clockwise This will ensure needle valve depression toll free 800 272 2775 phone 508 893 8999 web www btxonline com Volume Calibration Chart FORMULA Volume in Nanoliters 17952 x tip I D in micron 3 x Pressure in psi x Time in sec 5 EXAMPLE Z Volume 17952 x 5 3 X 10 X 1 Nanol
23. taper to the tip The smaller this angle the longer the tapered region The larger the tip the more material is delivered for the same applied pressure and time Just a 10 decrease in diameter decreases the delivery rate by over 30 A 10 decrease in taper angle longer taper would decrease the delivery rate about 10 The extreme sensitivity of delivery rate on tip diameter makes it important to have a reproducible pipette puller If you use published tip sizes as a starting point dis tinguish between the relevant inside diameter and the more visible outside diameter The ratio of the two is the same at the tip as for the original capillary glass toll free 800 272 2775 phone 508 893 8999 web www btxonline com BTX Harvard Apparatus MicroJect 1000A Pico Injector User s Manual Preliminaries Micropipettes continued 2 Choosing the Right Pipette Choosing a pipette size and shape for intracellular injection is difficult Larger tips deliver more material but increase the risk of cell damage caused by leakage around the pipette while in the cell or later by incomplete sealing The smaller the cell the smaller the pipette tip it will tolerate The smaller the tip the more likely it is to clog For reference intracellular electrophysiologists routinely record for an hour or so from cells of 10 micron diameter with pipette tips of 0 1 micron inside diameter Larger tips can therefore be used for brief injection in such cells For nuc
24. tion or the micropipette inside tip diameter or taper The dependence on net injection pressure duration and pipette taper angle is linear the dependence on tip diameter is cubic toll free 800 272 2775 phone 508 893 8999 web www btxonline com BTX Harvard Apparatus MicroJect 1000A Pico Injector User s Manual 14 General Information Micro Injection Substances Cell Types A wide range of substances have been micro injected onto or into a large number of cell types References 10 17 on page 16 give some recent examples Setting Pressures and Time Pulses 1 Balance Pressure Set the balance pressure while viewing the pipette under magnification This method will help the user to stabilize the solution within the pipette easily 2 Injection Pressure and Time Pulse Setting the initial injection pressure low prevents the loss of solution To easily obtain the desired pressure setting set the time pulse on 1 one second with the injection pressure set at its minimum Trigger the time pulse while viewing under magnification Increase the injection pressure until the solution within the pipette begins to flow out the tip opening The pressure shown on the LCD can now be used as the initial injection pressure setting Adjust the injection pressure and timing to obtain the desired injection 3 Holding Pressure Set the holding pressure at the minimum This will prevent possible damage to the cell Fill 3 4 of the pipette s ca
25. ume desired gets smaller the relative inflow gets larger even faster due to the smaller tip size being used To avoid this problem set the balance pressure before placing the pipette in the cell s external medium Ten percent of the injection pressure is a good starting value Exact balance is difficult to determine often the fastest way to handle this is to set the bal ance high enough that slight outflow is observed Balance is assessed by watching the movement of the liquid meniscus in the pipette The outflow left is still small com pared to the continuous streaming used in the continuous ejection technique If a filling fiber is used the capillary inflow is larger so a higher balance pressure is needed In general however when a balance pressure is being used such a fiber should not be used sometimes the injection material will run out spontaneously or the pressure needed to balance will abruptly change with time These may be due to particles lodging on the fiber toll free 800 272 2775 phone 508 893 8999 web www btxonline com BTX Harvard Apparatus MicroJect 1000A Pico Injector User s Manual 13 Using the MicroJect 1000A Pico Injector Balance continued If desired a rough estimate of the volume coming in can be made Focus the microscope on the liquid meniscus in the loaded pipette while its tip is still in air with the balance pressure set to zero Use an eyepiece reticle to measure the meniscus movement wh
26. upply MAKE PROPER CONNECTIONS Make sure all conections are made properly and securely ORIENT EQUIPMENT PROPERLY Do not position the equipment such that it is difficult to reach the disconnecting device OBSERVE ALL TERMINAL RATINGS Review the operating manual to learn the ratings on all connections AVOID EXPOSED CIRCUITRY Do not touch any electric circuitry inside the product DO NOT OPERATE WITH SUSPECTED FAILURES If damage is suspected on or to the product do not operate the product Contact qualified service personnel to perform inspection OBSERVE ALL WARNING LABELS ON PRODUCT Read all labels on product to ensure proper usage CAUTION Protective Ground Refer to Manual Terminal CAUTION CAUTION This product is not registered with the FDA and is not for clinical use on human or veterinary patients It is intended for research use only FOR RESEARCH USE ONLY NOT FOR CLINICAL USE ON PATIENTS toll free 800 272 2775 phone 508 893 8999 web www btxonline com BTX Harvard Apparatus MicroJect 1000A Pico Injector User s Manual Specifications MicroJect 1000A Pico Injector Specifications Input Gas Pressure 70 105 psi 480 720 kPa Injection Pressure 0 2 60 0 psi 413 kPa regulated multi turn control Balance Pressure 0 1 9 9 psi 68 9 kPa regulated multi turn control Fill Pressure 0 0 12 psi 82 kPa Vacuum Hold Pressure 0 to 1 25 kPa 12 7 cm of water Vacuum Clearing Press
27. ure Inlet pressure unregulated Injection Time 0 01 99 sec 1 99 sec Pressure Display Resolution 3 5 digits 0 1 PSI or IKPA Injection Count Display 0 9999 Duration Mode Internally timed or externally gated Trigger Mode Front Panel Foot Switch external TTL pulse BNC Pressure Monitor Out BNC Connector 10 mV psi or ImV KPa AC Power Lines External 9V DC 400mA min Power Supply universal input 90 to 264 VAC Accessories Supplied Input hose output hose holding hose and power module Weight 12 pounds 5 44 kg Dimensions H x W x D 3 5 in x 17 in x 9 8 in 89 mm x 432 mm x 250 mm Optional Accessories Foot Switch es up to four functions Pipette Holder up to 2 Input Hose Adaptor Warranty Two year parts and labor toll free 800 272 2775 phone 508 893 8999 web www btxonline com BTX Harvard Apparatus MicroJect 1000A Pico Injector User s Manual Introduction The MicroJect 1000A Pico Injector allows small liquid volumes to be delivered precisely through micropipettes by applying a regulated pressure for a digitally set period of time The pressure is applied pneumatically compressed gas to deliver volumes from microliters to femtoliters from the same instrument This digital injection pressure is enhanced by three auxiliary pressures FILL Suction can be applied to the delivery pipette to fill it from the tip HOLD A second internal vacuum pump is available to hold a suspended cell for injection with a second holding p
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