Nanoject II Auto-Nanoliter Injector

Video – Removing Wire Plunger

Video – Replacing Wire Plunger

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 micropipettes.

Contents:

• a. Injector head with cord.
• b. Control box.
• c. Power supply with cord.
• d. Capillary glass; 2 vials of 100 pieces per vial:
• 1. ( 3.5″ capillaries, 100 pcs. (Cat. No. 3-000-203-G/X)
  2. (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 pipettes. Cat. No. 3-000-027
• h. 1 Universal Adaptor

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 micropipettes pulled from capillaries with other dimensions will work. Never attempt to use micropipettes pulled from filament glass. Damage to the wire plunger will result and injection volumes will not be accurate.

Micropipette Pulling and Backfilling

The Nanoject II requires the use of pulled micropipettes 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 28g x 2″ needle and a syringe. Disposable spinal needles are also frequently used.

NOTE: THE NANOJECT II WILL NOT OPERATE PROPERLY WITHOUT BACKFILLING THE MICROPIPETTE.

Securing the Micropipette to the Injector Standard Collet/O-ring

Once the micropipette 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 micropipette 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 micropipette 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.

(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 PIPETTE.)

Filling the Micropipette

Once the micropipette 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 pipette 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 pipette into your sample and press and hold the “fill” button. The plunger will retract drawing with it the sample. The micropipette 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 micropipette. These bubbles can cause inaccurate injection volumes.

Injection

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.

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 micropipette 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.) can 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./second. Note: 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

Note: The dip switch settings are completely different from the previous model, Nanoject XV.

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

General Comments

1. 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.
2. The preferred tip size for Xenopus injection seems to be 20 microns. Some users report using 30 micron tips with no increase in mortality.
3. Injections of 25 nL or less generally require smaller tips, normally 10 microns.
4. A broken tip seems preferable for oocyte injection. Sharp polished tips tend to “skip”.
5. Virtually any oil appears suitable for backfilling.
6. A reported technique positions a drop of mercury between the oil and the sample in the micropipette. This apparently acts as a brake allowing better control of injection rate.
7. 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

Procedure for Re-homing Wire Plunger

Home Position: When the wire plunger tip is flush with the end of the black collet.

Before unplugging the unit from the power, it is essential that the wire plunger is returned to this position. Simply retract the plunger using the ‘fill’ button.

Why? Because when the unit is plugged in the next time, it will assume the wire plunger is in the home position. This position minimizes accidental damage to the plunger and prevents over extension of the plunger.

Full plunger movement from the end of the collet to fully extended is 26-27mm. Extending the plunger further than this can result in damage to the motor. There is no sensor to determine when full extension has been achieved; therefore, if the plunger is extended when first plugged in, over extension can result. If the plunger starts in the home position, it will not extend beyond 26-27mm.

Problem: Plunger is extended ½” when I plug the unit in.

Solution: Re-home the plunger.

How?:

(1.) Nanoject II (3-000-204/205A/206A)

• A. Connect injector head to control box.
• B. Plug-in power supply to wall outlet.
• C. Plug-in power supply connector to control box. A double beep will be heard.
• D. To reset plunger to the home position, push the ‘fill’ button and the inject’ button simultaneously. Hold both buttons until plunger retracts to the correct home position.
• E. Release both buttons.

To confirm unit is in the home position, follow “set-up check” described below.

(2.) Nanoject Variable/Fixed (3-000-203-XV & 3-000-203-X)

• A. Connect injector head to control box.
• B. Plug-in power supply to wall outlet.
• C. Plug-in power supply connector to control box. A double beep will be heard.
• D. Once in the correct position, unplug power supply connector from the control box.
• E. Wait 10 seconds, then plug-in again. A double beep will be heard.

To confirm unit is in the home position, follow “set-up check” described below.

Problem: Plunger wire is retracted and cannot be seen inside collet.

Solution: Extend plunger to the home position.

How?:

(1.) Nanoject II (3-000-204/205A/206A)

• A. Plug injector head to control box, power supply to outlet and control box.
• B. Press and hold ‘empty’ until plunger tip extends to the home position.
• C. Unplug power supply connector from the control box.
• C. Wait 10 seconds and reconnect power supply. A double beep will be heard.

To confirm unit is in the home position, follow “set-up check” described below.

(2.) Nanoject Variable/Fixed (3-000-203-XV/3-000-203-X)

• A. Plug injector head to control box, power supply to outlet and control box.
• B. Press and hold ‘empty’ until plunger tip extends to the home position.
• C. Unplug power supply connector from control box.
• D. Wait 10 seconds and reconnect power supply. A double beep will be heard.

To confirm unit is in the home position, follow “set-up check” described below.

Set-up Check:

1. Unplug power supply connector from control box.
2. Wait 10 seconds and reconnect. A double beep will be heard.
3. Press and hold ‘empty’ until another double beep is heard.
4. Plunger should be fully extended.
5. Measure with a ruler and the plunger should be extended 26-27mm.
6. Retract plunger and it should stop at the home position.

DO NOT OPEN THE BACK OF THE INJECTOR HEAD.

DO NOT OPEN THE CONTROL BOX.

CALL US FIRST AND ELIMINATE ANY UNNECESSARY PROBLEMS.

If you still have difficulty re-homing your Nanoject, call Drummond at 1-800-523-7480 or 610-353-0200.