Setting up an experiment

Electrophysiology Experiment Setup (Video *Updated*)

1. Prior to starting the experiment, check to see if either waste flask must be emptied. If they are more than halfway full, empty them. Then start the air pump by flipping on the red switch that's hanging on the blue shelf (assuming that you are using Betty). You can find it by following the power cord upward from the vacuum pump.

2. Fill the plastic water cup halfway with DI water and place into the clamp holder. Draw water into the syringe and inject it into the glass capillary tube to start the flow. Quickly place the capillary tube in the water in the cup.

NOTE: Multiple attempts might be necessary to push the water through and get the flow started.

3. If needed, remove air bubbles from the experimental chamber by shooting water from the “no-tape” pipette into the chamber. To get a good view, you will need to turn on the lamp and use the microscope. The idea is to pop/wash out the air bubbles. Check the long horizontal channels passing through the wells for bubbles. Make sure you completely fill your pipette with water by flushing the pipette up and down in the water.

5. Pinch the tubing to stop the flow of water and switch to Frog Ringer solution. Fill the reference well just short of full of KCl using the “red-tape” plastic pipette, using the KCl that's inside the red-taped cup from where you pulled the salt bridge from. Using the metal forceps, place the salt bridge to connect the reference well with the left chamber.

6. [DO ONLY IF TRAINED] Place the oocyte in the middle well of the experimental chamber with the yellow hemisphere up.

4. [DO ONLY IF TRAINED] Fill short glass pipettes with enough 3 M KCl to allow for good contact with the Ag/AgCl wire of the electrodes. The electrodes should be about half-way submerged in KCl. Avoid creating air bubbles and do NOT overfill. Insert glass pipettes into the electrodes and ensure the wire is in the KCl.

NOTE: Please use this link when putting together electrodes for an electrophysiology experiment

NOTE: If unsure about the presence of air bubbles, use the microscope to check for air bubbles in the tips of the glass pipettes. If you find air bubbles, flick the glass pipettes until they float up to the surface.

7. Put the pipette holders into the pipette holder boxes. Turn on the amplifier. For the window rig, this is the gray box with blue and black knobs, in the middle of the blue shelf, left of the rig itself. Turn on the oscilloscope. This is above the amplifier, and you do this by clicking the black square button that has "power" above. The red right above that will glow once it's on.

8. Move the glass pipettes into the solution (breaching the surface) and position them close to the oocyte. Zero the voltage of channels 1 and 2 by turning the offset knobs.

NOTE: Clockwise makes the voltage more positive; counterclockwise makes it more negative.

9. [DO ONLY IF TRAINED] Place the glass pipettes on the oocyte at about a 45° angle with respect to the chamber. You should see 2 dimples form. Softly tap either the upper or lower adjustment knob of the micromanipulators to gently penetrate the oocyte. You have succeeded if the voltage on channels 1 and 2 drops to a negative value, usually between -20 mV and -30 mV. If there is no change, continue tapping until the oocyte is impaled. Channel 1 measures from the left electrode and channel 2 measures from the right electrode.

NOTE: If the voltage hasn't changed, and you're sure that the oocyte has been impaled, there could be a connectivity issue within the electrodes. Please remove them from the rig and make sure all of the parts are screwed in tight, that the metal rod is only partially submerged in KCl, and that there are no air bubbles in the glass pipette.

10. Turn on the compute, using the login information that's written on the pale pink tape on the monitor. Open Clampex. Under File, click Set Data File Names. Create a new folder and name it after today’s date. If one is already created, then skip this step.

11. Open that folder and name the file using your first and last initials followed by the date without dashes or spaces and including zeroes (i.e. mjc110616). Click on the number box to the right of the file name and change it to 0001. If this is your second experiment of the day, insert a b between your initials and the date