Junior Patcher Checklist

This checklist may help when you are only learning how to patch. There are so many little actions that all need to be done in the correct order that people get confused. No wonder! So just follow this simplified plan, point by point, and it should be fine.

1. Fill new pipette to 1/3 – 1/2 of its length
2. With a kimwipe (tightly rolled into a resemblance of a paper-needle) remove excess liquid from the back of the pippette
3. Get rid of bubbles in the tip (through knocking the pipette gently, while holding it upright)
4. Install the pipette into the holder
5. Apply positive pressure (<0.1 ml of air)
6. Put the amplifier in Voltage Clamp at 0 mV.
7. Go down until the tip touches the liquid (manipulators should be in the "high speed", or "bunny" mode)
8. Measure pipette resistance (should be 5-15 MΩ; do it in "Bath" mode).
9. Focus on the tip, ensure that there are no bubbles or junk inside (or at least not too much junk)
   1. If any bubbles are detected, blow them through:
   2. inject air – lock – draw – inject … ; repeat it 3 times; as soon as the bubble goes through – release immediately.
   3. Don't forget to re-inject a tiny bit of positive pressure after you are done.
   4. If the tip has junk outside – change the pipette; it's not gonna work.
10. Focus lower than the tip, then move the tip to the focus plane. Repeat until slightly above the brain (when the brain is visible, but is still blurry).
11. Switch the manipulator to low speed ("turtle" mode)
12. Change the objective from 10x to 60x
13. Find the tip (normally you would have to focus down, as 60x is focused slightly above the 10x).
14. Lower the tip to just above the cell layer (similar to p. 10, but not in fine mode for both the tip and the microscope)
15. Choose a cell
16. Cancel pipette offset & pipette capacitance
17. Patch:
    1. Place the tip exactly above the point between the center and the side of the cell
    2. Focus down at the cell, but slightly above its midline
    3. Move pipette down until it is near the cell and a darker dimple can be seen
    4. Release positive pressure (by opening the valve)
    5. Gently apply suction – until gigaseal is formed
    6. Put the amplifier in Voltage Clamp at -60 mV
    7. Switch from "Bath" to "Seal" mode. Apply measured suction until you break into the cell.
18. Measure cell parameters (in "Cell" mode), and follow the protocols.