Patch-Pipettes EPC9 Manual 72
Hard-glass pipettes often have a narrow shank after pulling and consequently a
higher resistance. Hard glasses tend to have better noise and relaxation properties,
however: the important parameter here is the dielectric loss parameter, which
describes the AC conductivity of the glass. Although the DC conductivity of most
glasses is very low, soft glasses in particular have a conductivity around 1 kHz; that
is sufficiently high to become the major source of thermal noise in a patch-clamp
recording. We find that Kimax glass is a good compromise for whole-cell recording.
Borosilicate and, especially, aluminosilicate glasses (Rae and Levis, 1984) have low
dielectric loss and are desirable for the lowest-noise recordings. They do not
necessarily form the best seals, however; this might be due to evaporation of metal
onto the glass surface during the high-temperature pulling and polishing steps.
Pulling
Pipettes are pulled in two stages: the first to thin the glass to 200-400 µm at the
narrowest point over a 7-10 mm region, and the second to pull the two halves apart,
leaving clean, symmetrical breaks. Both halves can be used. The length of the first
pull and the heat of the second pull are the main determinants of the tip diameter of
the final pipette.
A number of commercial pullers can be used to make pipettes. For reproducibility,
however, a regulated current supply to the heater coil is best. A mechanical stop to
set the length of the first pull is also important for reproducibility.
Coating
The capacitance between the pipette interior and the bath, and also the noise from
dielectric loss in the glass, can be reduced by coating the pipette with an insulating
agent such as Sylgard (Dow Corning Corp., Midland, MI, U.S.A.). Sylgard is pre-cured
by mixing the resin and catalyst oil and allowing it to sit at room temperature for
several hours (or in an oven at 50 ˚C for 20 min) until it begins to thicken. It can then
be stored at -18 ˚C for many weeks until use. The Sylgard is applied around the lower
few mm of the electrode to within 10-20 µm of the tip and then rapidly cured by a
hot-air jet or coil. Coating should be done before the final heat-polishing of the
pipette, so that the heat can evaporate or burn off any residue left from the coating
process.
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