Axon Axopatch 200B, Theory Of Operation

Page: 47 / 153

USE OF THE PATCH CLAMP – A TUTORIAL • 31
Chapter 3
electrode and the patch electrode will show up as a non-zero tracking voltage on the meter.
Adjust the PIPETTE OFFSET potentiometer until V
TRACK
is zero. (Some investigators
prefer not to use the tracking circuit but rather do their early adjustments and seal
formation in voltage clamp mode. If you prefer this approach, place the MODE switch in
the V-CLAMP position and the meter switch to I before placing the pipette tip into the
bath. With the tip in the bath, adjust the PIPETTE OFFSET control until the current on the
meter reads zero.)
Note: This is a good point to check the stability of your bath (ground) and patch
(recording) electrodes. Drifting electrodes will cause a constantly changing V
TRACK
voltage in TRACK mode whereas in V-CLAMP the current will continually drift
off zero.
Switch the external command switch to the SEAL TEST position. A 5 mV positive-going
rectangular pulse is applied to the patch clamp input. The pulse frequency is the same as
the AC line frequency and the duty cycle is 50%. This will result in a square pulse of
current whose amplitude depends on the pipette resistance as shown in Figure 7.
5 nA
1 nA
100 pA
1 M
10 M
100 M
PIPETTE
RESISTANCE
(ohms)
CURRENT PULSE
Figure 7. Pipette resistance measurement using seal test.
Lower the pipette until it just touches the cell. You will be able to tell when this happens
because the resistance will increase as the pipette makes contact with the cell membrane,
causing a decrease in the size of the current pulse (Figure 8, three upper traces). Continue