Nanoject II/Auto Nanoliter Injector Operation and Service Manual
This equipment is ready for use as received. It does require pulled capillary tips. It is essential that only the capillaries supplied or exact replacement capillaries are used for pulling micropipets.
Contents:
a. Injector head with cord.
b. Control box.
c. Power supply with cord.
d. Capillary glass; 2 vials of 100 pieces per vial:
- ( 3.5" capillaries, 100 pcs. (Cat. No. 3-000-203-G/X))
- (7.0" capillaries, 100 pcs. (Cat. No. 3-000-203-G/XL))
e. Replacement O-ring kit with collet, standard. (Cat. No. 3-000-002-KN)
f. 1 Allen wrench for replacing wire plunger.
g. 1 30g x 2" needle for backfilling pipets. Cat. No. 3-000-027
Operation:
The injector head should be mounted in a suitable micromanipulator or stereotaxic unit. Marzhauser model MM33 is quite suitable and frequently used. The aluminum barrel with the Drummond logo, provides an ideal clamping surface. Its diameter measures 11mm.The precise operation of the Nanoject II depends greatly on the use of tips prepared from the glass provided. We cannot guarantee that glass micropipets pulled from capillaries with other dimensions will work. Never attempt to use micropipets pulled from filament glass. Damage to the wire plunger will result and injection volumes will not be accurate.
Micropipet Pulling and Backfilling
The Nanoject II requires the use of pulled micropipets from the glass provided. Ideally, the tip size should be 10-30 microns in size. The capillary glass provided is produced from N-51-A material and has a softening point of 780º C. Many researchers pull the tips and then break them off with forcepts. This enables piercing the cell membrane much easier with no skipping.Once the tips are pulled, they must be "backfilled" with oil(or other non- compressible fluid) before attachment to the injector. Silicone or mineral oil is frequently used. Backfilling is facilitated by using the 30g x 2" needle and a syringe. Disposable spinal needles are also frequently used.
NOTE: THE NANOJECT II WILL NOT OPERATE PROPERLY WITHOUT BACKFILLING THE MICROPIPET.
Securing the Micropipet to the InjectorStandard Collet/O-ring
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Once the micropipet is backfilled, loosen the collet. The pointed wire plunger should be positioned so you can just see the tip flush with the end of the collet (slightly recessed is also acceptable). This is referred to as the "home" position. Push the micropipet onto the wire plunger and as you push the tip on, feel it go through the large O-ring and seat in the white spacer. Once positioned, tighten the collet securely. Give the micropipet a pull to confirm it is securely mounted. See fig. 1 for proper configuration of the O-rings and the white spacer. It is absolutely essential that these components are properly configured.
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(NOTE: THE WHITE SPACER HAS ONE FLAT SIDE AND ONE SIDE WITH A RECESS MACHINED AROUND THE HOLE. THIS RECESS IS TO RECEIVE THE BACK END OF THE PIPET.}
Optional Acessories
Special Collet/O-ring (3-000-002-K4) for use with flared glass (3-000-204-G/X).
This optional O-ring kit is available for use with the optional flared cappillary tubes.The unit is supplied in a separate marked vial for use with the flared capillary tubes. These tubes are flared on both ends so two micropipets can be produced with each pull. The use of this collet requires removal of the collet supplied, removal of the large O-ring, and removal of the white spacer. The small "sealing" O-ring can be used (2 additional "sealing" O-rings are supplied in the vial for future replacement use). Also included is a "pressure sleeve" which will place pressure against the flare and compress it into the sealing O-ring for a secure fit.
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The use of this collet and the slow injection speed may enable you to pull smaller tips. One suggestion would be to break the tip off after you push it through the pressure sleeve and collet. Once the unit is placed on the wire plunger, tighten the collet securely.
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Filling the Micropipet
Once the micropipet is secured to the collet, press and hold the "empty" button. This will drive the wire plunger out forcing oil to the tip of the pipet and any excess oil will be expelled. Hold the empty" button until an audible beep is heard. The plunger is now fully extended (approx. 27mm from the end of the collet).Now place the tip of the pipet into your sample and press and hold the "fill" button. The plunger will retract drawing with it the sample. The micropipet when fully filled, will contain approximately 5uL of sample. At any time you can stop filling by letting go of the "fill" button. Continued filling can be accomplished by pressing the button again. Viscous samples may require you to fill in small steps allowing the sample to equilibrate in the tip before continued filling. Do not allow air bubbles to form in the micropipet. These bubbles can cause inaccurate injection volumes.
Injection
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Injection volumes are determined by the position of the dip switches. Only dip switches #1- #4 control the volume see fig. 3). Dip switch #5 controls the injection rate and the fill rate.
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Once the volume has been selected, each time the "inject" button is depressed an audible beep will be heard and the selected volume will be dispensed. The actual injection of the micropipet into the oocyte or tissue is produced by the micromanipulator.Multiple injections can be made in one location by simply pressing the "inject" again. Pressing "inject" again before the first injection is complete will not produce a second injection. Listen for the beep.
The control buttons work as follows:
FILL Retracts wire plunger while position is depressed. "Beep" will sound at extreme fill position.Dip switch #5 will control the rate of filling.#5 UP = 46 nL./second fill rate#5 Down = 23 nL./second fill rate EMPTY Extends wire plunger while position is depressed."Beep" will sound at fully extended position(approx. 27mm from end of collet). Empty speed is approximately 92nL/second. Fast empty (230 nL./sec.) be achieved by holding the empty button and pressing the "fill" button once. The plunger will maintain the faster speed until the empty button is released. The unit will always start in the slower speed.
FAST FILL & FAST EMPTY
Dip switch #5 does not have any effect on the empty mode.To empty fast, hold down the empty button and touch the fill button one time. The empty speed will increase to 230 nL./second.The fill mode also has a fast speed. Hold down the fill button and touch the empty button one time. Depending on the position of dip switch #5 the fast rate will vary.#5 UP = 230 nL./second#5 DOWN = 92 nL./secondNote: The unit will always start in the slower speed.
Setting the Injection Volume and Injection Speed
Injection volumes and injection speeds are controlled by the positions of the dip switches located on the side of the control box. DIP SWITCH
| VOLUME |
1
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2
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3
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4
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5 INJECTION RATES
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| 2.3 nL. |
U
|
U
|
U
|
U
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U FAST = 46 nL/sec.
|
| 4.6 nL. |
D
|
U
|
U
|
U
|
|
| 9.2 nL. |
U |
D |
U |
U |
D SLOW = 23 nL/sec. |
| 13.8 nL. |
D |
D |
U |
U |
|
| 18.4 nL. |
U |
U |
D |
U |
. |
| 23.0 nL. |
D |
U |
D |
U |
|
| 27.6 nL |
U |
D |
D |
U |
. |
| 32.2 nL. |
D |
D |
D |
U |
|
| 36.8 nL. |
U |
U |
U |
D |
. |
| 41.4 nL. |
D |
U |
U |
D |
|
| 46.0 nL. |
U |
D |
U |
D |
|
| 50.6 nL. |
D |
D |
U |
D |
Note: The dip switch settings are completely different from the previous model, Nanoject XV.
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| 55.2 nL. |
U |
U |
D |
D |
| 59.8 nL. |
D |
U |
D |
D |
| 64.4 nL. |
U |
D |
D |
D |
| 69.0 nL. |
D |
D |
D |
D |
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fig. 3
Cleaning Recommendations
The injector can be cleaned by removing the collet, O-rings, spacer, etc. and wiping them with alcohol. Do not soak in liquid or autoclave the Nanoject II. The control box can be cleaned with a clean dry cloth.
Servicing the Unit
There are only two common service functions that the end user will perform. (1) Replacement of O-rings and (2) replacement of the wire plunger.
ANY ADDITIONAL SERVICE FUNCTIONS, SHOULD BE PERFORMED BY DRUMMOND SCIENTIFIC. UNAUTHORIZED TAMPERING CAN VOID WARRANTY. CONTACT DRUMMOND AT : 1-800-523-7480 OR 610-353-0200.
Replacement of O-rings
After a period of time or heavy use, some leakage of oil during injection might be observed. To correct this, the O-rings must be replaced. Two O-ring kits are provided. Refer to fig. 1 or 2 for proper installation. This step is critical for proper operation. Note the white spacer in fig. 1 must be properly oriented.
Replacement of wire plunger
To change the wire plunger (cat. no. 3-000-000-203-X) remove the collet and all collet components. Hold the motor housing and unscrew the aluminum barrel. Once free, pull off. You will see a brass fitting with two Allen screws. Unscrew these with the wrench provided. Remove old plunger and place new one in fitting. Tighten the screws making sure wire is seated fully and straight. Replace aluminum barrel and collet.
Plunger in "HOME" position
When you plug in the Nanoject II, it will assume it is in the "home" position. When in this position, the tip of the wire plunger should be slightly recessed from the end of the collet. This prevents accidental damage to the plunger when not in use. To reset the plunger to this position, push the fill to retract or the empty to extend the plunger and simultaneously hold the inject button. When in the home position, release both buttons and the unit is reset.When unplugging the unit, make sure the plunger is in the home position. Upon restarting the unit, it will start in this position.
Optional Accessories
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Catalog No.
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Name
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Description
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3-000-026
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Foot Switch
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An optional foot switch is available that plugs into the control box that enables you to inject using the foot switch.
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3-000-024-A
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Nanoject Universal
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Enables use with most micromanipulators.
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General Comments
- The wire plungers on all injectors have a point on one end. This wire can be reversed if a blunt end is desired. The point should allow bubbles in the oil to escape when backfilling tips.
- The preferred tip size for Xenopus injection seems to be 20 microns. Some users report using 30 micron tips with no increase in mortality.
- Injections of 25 nL or less generally require smaller tips, normally 10 microns.
- A broken tip seems preferable for oocyte injection. Sharp polished tips tend to "skip".
- Virtually any oil appears suitable for backfilling.
- A reported technique positions a drop of mercury between the oil and the sample in the micropipet. This apparently acts as a brake allowing better control of injection rate.
- An excellent reference to all procedures involving the Xenopus oocyte is:
Kay, B. and H. Peng. "Xenopus laevis: Practical Uses in Cell and Molecular Biology". Methods in Cell Biology. Vol. 36. Academic Press* Back to Nanoject II
* Nanoject II Micropipette Holder
* Nanoject II Specifications and Parts
