Nanoject II/Auto Nanolitre Injector Operation and Service Manual

This equipment is ready for use as received. It requires pulled capillary tips and 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:
(3.5" capillaries, Pk 100 (Cat. No. 3-000-203-G/X))
(7.0" capillaries, Pk 100 (Cat. No. 3-000-203-G/XL))
e. Replacement O-ring kit with collet, standard. (Cat. No. 3-000-002-KN)
f. One Allen key for replacing wire plunger.
g.One 30g x 2" needle for backfilling pipettes. Cat. No. 3-000-027
h. Universal Adaptor (Cat. No. 3-000-024-A) -enables use with most micromanipulators.

Operation:
The injector head should be mounted in a suitable micromanipulator or stereotaxic unit. Marzhauser
model MM33 is quite suitable and frequently used. The aluminium 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 forceps.
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
MICROPIPETTE.

Securing the Micropipette to the InjectorStandard 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 PIPETTETE.)

Optional Acessories
Special Collet/O-ring (3-000-002-K4) for use with flared glass (3-000-204-G/X).
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This optional O-ring kit is available for use with the optional flared capillary 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 micropipettes 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.

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.

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 5µL of sample. At any time you can stop filling by releasing 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. 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.) 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.

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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

VOLUME 1 2 3 4 5 INJECTION RATES

2.3 nL. U U U U 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
55.2 nL. U U D D different from the previous model, Nanoject XV.
59.8 nL. D U D D
64.4 nL. U D D D
69.0 nL. D D D D

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 AN AUTHORISED
DRUMMOND SCIENTIFIC REPRESENTATIVE. UNAUTHORIZED TAMPERING CAN VOID
WARRANTY.

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 aluminium 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
aluminium 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

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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

Cat. No. Description Details

An optional foot switch is available that plugs into
3-000-026 Foot Switch the control box that enables you to inject using the
foot switch.

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 micropipette.
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

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