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Tutorial
To make your own syringe with thin tubing, remove the internal plunger. Place
your hands on opposite ends of the syringe and rotate it while heating its
midsection over a small flame. Once the plastic begins to melt, remove the
syringe from the flame and pull the ends apart. Gentle pulling will produce a
long, thin plastic tube. After cooling, cut this tube such that a 7 to 10 cm
section extends from the back end of the syringe. Reinsert the plunger and
make sure that the tube will allow solution to flow through it.
Mounting the Micropipette
When the micropipette is inserted into the holder, the Ag/AgCl wire need only
be immersed into the solution far enough for electrical contact to be made.
There is no reason to have the wire close to the tip of the micropipette. Loosen
the pipette cap of the holder. Feed the silver wire into the filled micropipette
and push the micropipette through the pipette cap into the barrel of the holder.
Carefully tighten the pipette cap so that the micropipette is held in place.
Attach the HL-U holder to the AP-1A headstage that is mounted on a
micromanipulator.
Positioning the Micropipette Tip
While the current is measured, the micropipette tip is slowly advanced against
the cell membrane (identical to a standard patch-clamp measurement at the
time of seal formation). As the pipette pushes against the cell and increases the
cleft resistance, the current falls as the sum of the pipette and cleft resistance
increases. A voltage divider forms by the resistances of micropipette tip and
the cleft between it and the cell. When resistance has gone up 33%, then the
current drops by 25%. This results in a voltage (V
m
) at the cell surface that is
about 25% of the commanded voltage. Presumably it is the voltage at this
location that is responsible for creating the lipidic channels across the cell
membrane through which genes, peptides, dyes, and most other compounds can
flow. Although higher degrees of indentation result in a larger fraction of the
pipette voltage being applied at the cell surface, there is presumably a trade off
between the increased effectiveness and damage to the cell. It remains to be
Axoporator 800A Theory and Operation, Copyright 2005 Axon Instruments / Molecular Devices, Corp.
