- setup amplifier
- in voltage clamp dial in holding voltage to ~-50mV
- turn switch to off — which switch??
- turn on seal test
- fill patch electrode with internal saline ??
- make sure not clogged or have any bubbles in tip
- put electrode in bath
- make sure pipette resistance is acceptable (8-12 mega Ohms)
- apply positive pressure before it hits the bath!
- slowly lower pipette toward cell of interest
- once close to cell set speed to 5
- switch to mouth pressure
- approach cell with pipette
- position pipette tip over cell
- slowly lower tip
- cell should dimple from positive pressure
- release positive pressure
- cell should rebound up to the pipette
- oscilloscope should change from steps to transients as seal is formed
- wait about a minute to make sure the seal is stable and that the pipette isnt drifting
- cancel capacitance transients with fast pipette capacitance
- set oscilloscope scale to 100mV to dial in compensation??
- turn on voltage command by flipping the switch from off to negative
- break into the cell with snappy suctions that slowly increase in intensity
- you have broken into cell when capacitance transients come back
- acquire trace for records - tells you how good access is
- turn off seal test and flip to I=0 mode
- aquire another trace
- this gives good sense of overall quality of recording
- ideally sitting at hyperpolarized potential (-30-55 is good for most cells)
- spontaneous behavior shouldnt be too crazy
- if things look off, hyperpolarizing current injection might fix everything
- remove all voltage dialed in before starting experiment
- turn holding command knob to 0
- flip to current clamp normal
- dial in a bit of holding current to help cell sit at a measureable potential
- most cells need a few pA of negative current
- acquire a few more traces
- begin experiment
references
typed up version of hand written notes in tuthill lab documents
google doc scan of original