Patch-Pipettes EPC9 Manual 73
Heat Polishing
Heat polishing is used to smooth the edges of the pipette tip and remove any
contaminants left on the tip from coating. It is done in a microforge or similar setup
in which the pipette tip can be observed at a magnification of 400-800x. The heat
source is typically a platinum or platinum-iridium wire; to avoid metal evaporation
onto the pipette, the filament is coated with glass at the point where the pipette will
approach it. To produce a steep temperature gradient near the filament (which helps
make the pipette tip sharply convergent), an airstream can be directed at the
filament. The amount of current to pass through the filament must be determined
empirically for each type of glass, but a good place to start is with sufficient current
to get the filament barely glowing. The typical practice is to turn on the filament
current and move the filament toward the pipette (which, being stationary, should
remain in focus). Since the opening in the pipette tip is usually at the limit of
resolution of viewing, you might not see the change in shape at the tip, but instead
only a darkening of the tip. You can tell whether you have melted the tip closed, and
also get an idea of the tip diameter, by blowing air bubbles in methanol with air
pressure supplied by a small syringe.
Use of Pipettes
Pipettes should be used within 2-3 hours after fabrication, even if stored in a covered
container; small dust particles from the air stick readily to the glass and can prevent
sealing. However, with some easy-sealing cells we have made the experience that
pipettes may even be used the next day. It is very important to filter the filling
solutions (e.g., using a 0.2 µm syringe filter). Pipettes can be filled by sucking up a
small amount of solution through the tip. This can be done by capillary force (simply
dipping the tip for a few seconds in a beaker containing the pipette solution), or by
applying negative pressure at the back of the pipette (e.g., using a 5 ml syringe).
Thereafter, the pipette is back-filled; the pipette should only be partially filled, just
far enough to make reasonable contact with the electrode wire (the pipette holder is
not filled with solution, but is left dry). Overfilling the pipette has disastrous
consequences for background noise because the solution can spill into the holder,
wetting its internal surfaces with films that introduce thermal noise. Bubbles left in
the pipette from filling can be removed by tapping the side of the pipette.
For low-noise recording, the electrode holder should be cleaned before each
experiment with a methanol flush, followed by drying with a nitrogen jet. Before you
insert a pipette into the holder, it is a good idea to touch a hand to a metal surface of
the setup to discharge any static electricity that you may have picked up. Be sure to
tighten the holder firmly enough that the pipette does not move (on a scale of 1 µm)
when you give suction. Then, when you change pipettes during an experiment,
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