Using the Patch Clamp
EPC9 Manual
75
10. Using the Patch Clamp
We give here a brief description of the techniques for establishing a seal and
recording from a membrane patch or from an entire small cell.
Forming a Seal
Initial Setup
The object is to apply voltage pulses to the pipette and observe the
Current Monitor
signal on an oscilloscope to monitor the pipette resistance. A convenient way to set
the desired adjustments of the amplifier is by activating the
Set-Up
mode. The
approach to the cell membrane and the formation of a giga-seal cause the resistance
to increase, reducing the currents. A convenient pulse amplitude is 2 mV, which can
be obtained from the built-in pulse generator. The 2 mV pulses will cause 1 nA to
flow in a 2 M
Ω
pipette.
For observation of the current pulses, it is convenient to pick a
Gain
setting and
oscilloscope sensitivity such that the current through the open pipette is reasonably
sized. Other recommended settings are also preset in the
Set-Up
mode:
Voltage Clamp
and
V-membrane
= 0;
Filter 1
is set to 10 kHz and
Filter 2
to 3 kHz,
R
s
-comp
to
Off
,
C-
slow Range
to
Off
and
Stimulus Rise Time
to 20 µs. Before the pipette is inserted into the
bath, the current trace should be flat, except for very small capacitive pulses due to
the stray capacitance of the pipette and holder.
Entering the Bath
The surface of the solution is relatively “dirty”, even if (as we strongly recommend)
you aspirate some solution from the surface to suck off dust and contaminants. For
this reason it is important to apply a small amount of positive pressure to the pipette
before you move its tip into the bath, and also to avoid going through the air-water
interface more than once before forming a seal. When you do move the pipette tip
into the bath, the current trace may go off-scale (check clipping); in that case, click on
the
Auto-V
0
button or reduce the gain until the trace reappears. From the size of the
current response to the test pulses, the pipette resistance can be calculated (the
pipette resistance is displayed in the EPC9 window as
R-membrane
). If there should
be no change in the trace upon entering the bath, check for an open circuit, for
