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3.22 General Procedure for TEVC
1.Oocytes have been injected a few days earlier and are ready for voltage clamp (see injection protocol above).
2.Backfill two pipettes with 3 M KCl solution using a fine syringe needle (that can fit into the 0.78 mm ID of your pipettes) and place them in a storage box (away from the dust).
3.Place one electrode into each of the two holders and insert the holders into the two headstages of your amplifier (see Note 61).
4.Using the coarse manipulators, immerse the tips of your electrodes into the recording bath solution. One should read about 1 MΩ for each microelectrode. Read the values following the procedure of the amplifier you are using (see Note 62).
5.Look through the compound binocular microscope to visualize the tips.
6.Zero the pipette offset for both electrodes (see Note 63).
7.Impale the oocytes successively with both microelectrodes. The DC meter of the amplifier will read the resting membrane potential for electrode P1 (V1) and P2 (V2) (see Note 64).
8.Reset your clock and enter all relevant parameters in your logbook.
9.You are now ready to record whole-cell currents from the injected oocytes. Follow the instructions for your particular amplifier (see Note 65) (Fig. 1).
You may start by reading The Axon Guide (download from Molecular Devices website). There you can find a detailed description of the method and your hardware. It was very helpful when I did TEVC during my postdoctoral training. You should connect your pieces of equipment according to your task (nothing really extraordinary). Axoclamp requires a special kind of grounding described in the book. You can test your system with a TEVC model cell supplied with the amplifier.