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3.5.5
Technical Tips for Pulling Micropipettes
3.5.5.1
Regulating the Time it takes to pull a Sharp Pipette
HEAT. If the pull takes longer than eight seconds while using 1.0mm O.D. tubing to pull a
fine- micropipette, increase the HEAT. To do this in a methodical fashion, increase the HEAT
value in five unit increments, each time monitoring pull time until the pull takes place in less
than eight seconds.
If the pull occurs in less than three seconds after you start, decrease the HEAT value in a
similar fashion.
For 2mm O.D. tubing, the pull should occur between 10 and 15 seconds after the start. Make
corrections as outlined above for smaller tubing.
3.5.5.1.1
Pipette Position
The position of the glass within the filament will also affect the time it takes to pull a pipette.
When using a trough filament the glass should be about 0.5mm above the bottom of the
filament and centered front to back. In the case of a box filament the glass should be in the
center of the filament. Filament positioning is covered in the next section of this manual
“Heating Filaments”.
3.5.5.2
Regulating the Length and Tip Size of a Sharp Pipette
HEAT. Higher HEAT settings will give longer and finer tips. A HEAT value equal to the
Ramp Test value plus 10 units will generally give a very fine tip.
NOTE: At high HEAT settings (filament white-hot), the filament life is greatly reduced.
Initially, use a setting equal to the ramp value plus 5: electrode length is controlled by air
pressure adjustments. If this is insufficient, a wider or more narrow filament can be
installed.
3.5.5.2.1
Filament Width
Filaments narrower than 2mm cannot form as fine a tip as the wider filaments. The tip size
will decrease with increasing filament width until a width of 3mm is reached. Increasing the
filament width beyond 3mm will produce longer and more gradual tapers (which may
penetrate better in some cases). The tip, however, will not be any smaller.
3.5.5.2.2
Air Flow
In general, electrodes will not be formed if the air pressure is set too high. It is thus
recommended that the pressure be set to standardized values of 500 for thick walled glass
and 300 for thin walled tubing. However, as outlined above, under
Micropipette/microinjection needle fabrication, the length of pipette tips can be controlled by
varying air pressure. Furthermore, when making patch pipettes, if increasing TIME to its
maximal setting of 255 does not provide enough cooling to produce tips with a short enough
taper, then using the delay mode or increasing pressure above the standard values may be
warranted.
3.5.5.2.3
DELAY Mode of Active Cooling
As discussed previously this manual, switching from the TIME to the DELAY mode of active
cooling may give more precise, and a wider range, of control over the length (or taper) of a
sharp pipette tip. The delay mode is often employed when using thick walled glass or for
programs designed for the fabrication of pronuclear injection needles.
P-97 FLAMING/BROWN MICROPIPETTE PULLER OPERATION MANUAL – REV. 2.43 - DOM (20161118)
Summary of Contents for P-97
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