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A Procedure for Recording from Oocytes
Current Electrode Placement
7)
Advance the current electrode until the tip is in the chamber bath solution.
Adjust V
OFFSET for a zero reading on the CURRENT ELECTRODE METER.
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This will establish a null reference allowing the resting potential to be
directly read.
8)
With the CLAMP SELECTOR switch in the off position, the resistance of the
CURRENT ELECTRODE pipette is tested in the same manner as the VOLTAGE
ELECTRODE. Pressing the V
to be passed across the CURRENT ELECTRODE. The resulting voltage (in mV) will
be displayed on the METER in the BATH ELECTRODE section. From that value, the
resistance of the pipette can be calculated exactly as described above (i.e., divide
the readout by 10 to get the resistance in MΩ). Since the current electrode has a
larger diameter, its resistance should be less than that of the voltage electrode
(about 1.0 MΩ or less).
9)
If no voltage display is present during the electrode test procedure and you're
sure that the electrode is contacting the bath, perform the following checks:
a) Make sure that all cables are connected properly.
b) Check to see that the aluminum shield around the current
electrode pipette (if used) is not touching the bath solution or
the electrode wire.
c) Check the current electrode to see if there is a bubble in the pipette causing
an open circuit.
Impaling the Cell
10) Recheck the VOLTAGE ELECTRODE METER to verify that the potential is correctly
offset to read 0.0 mV and readjust the Vm OFFSET control if needed.
11) Advance the VOLTAGE ELECTRODE until its tip is slightly depressing the plasma
membrane of the cell and depress the Vm BUZZ pushbutton. This will produce a
1 V, 1 kHz oscillation at the voltage electrode, disrupting the membrane and
causing the tip of the electrode to impale the cell with no further movement of
the micro-manipulator (this technique will work best with "fresh" oocytes, i.e.,
1 or 2 days post-excision). If the buzz technique fails to cause electrode
penetration, further advance the voltage electrode until it "pops" through the
membrane.
12) The potential across the membrane will now be displayed on the VOLTAGE
ELECTRODE METER.
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ELECTRODE TEST pushbutton will cause a 10 nA current
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Publication 5720-001 REV1.0
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