Using the Patch Clamp EPC9 Manual 76
example: 1. a bubble in the pipette; 2. faulty connection to the probe input; 3. bath
electrode not connected.
There is invariably a small offset potential between the pipette and bath electrodes.
The V
0
control is designed to provide a bucking potential to cancel this offset. With
the pipette in the bath, select the Auto-V
0
button.
Forming a Gigaseal
When the pipette is pushed against a cell, the current pulses will become slightly
smaller to reflect the increasing seal resistance; when the positive pressure is
released, the resistance usually increases further. Some cell types require more
“push” from the pipette than others, but an increase in resistance of 1.5 (i.e., a
reduction in the current pulses by this factor) is typical.
Application of gentle suction should increase the resistance further, and result
(sometimes gradually, over maybe 30 s; sometimes suddenly) in the formation of a
gigaseal, which is characterized by the current trace becoming essentially flat again
(hyperpolarizing the pipette to -40 to -60 mV often helps to speed the seal
formation). To verify gigaseal formation, increase the Gain to perhaps 50 mV/pA; the
trace should still appear essentially flat except for capacitive spikes at the start and
end of the voltage pulse.
Cell-Attached Recording
You can now use the On Cell mode, which will automatically adjust C-fast and τ-fast
controls to minimize the size of these spikes (some improvement can sometimes be
obtained by manual adjustment). Transient cancellation will be essential if you will
be giving voltage pulses in your experiment. It is a good idea to start out with V-
membrane set to zero, as we specified above; an alternative is to start with V-membrane
set to the holding potential you desire (e.g., -70 mV). If no voltage jumps are
required, turn the stimulus off to avoid introducing artifacts. If voltage jumps are to
be applied, switch the Gain and Filters to the values you will be using (the settings
may be programmed under On Cell).
Be sure to use Gain settings of 50 mV/pA or above for lower noise in single-channel
recordings. Keep the Filter 1 switch set at 10 kHz unless you actually will need the
full 60 kHz bandwidth for some reason; otherwise you might drive the current
monitor output or your recorder's input amplifiers into saturation with the very
large amount of high-frequency noise. Should you use the full bandwidth, you
should avoid gain settings above 100 mV/pA for the same reason.
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