Het maken van Patch-pipetten en Sharp elektroden met een programmeerbare Puller

Glass capillary pipettes have become ubiquitous in laboratories for a wide variety of techniques, such as single cell electroporation, DNA microinjection and patch clamp electrophysiology To prepare glass capillary patch clamp electrodes for recordings or silicate glass is pulled on a programmable polar. The electrode tips are coated with wax, and then the tips are fire polished and stored for future use. Hi, I'm Miriam Goodman from the Department of Molecular and Cellular Physiology at Stanford University.

In this video, I'll demonstrate how to make patch clamp pipettes and sharp electrodes for electrophysiology. Glass micro electrodes, also called pipettes, have been a workhorse of electrophysiology for decades. Today, these pipettes are made from glass capillaries using a programmable pull.

So let's get started. To make patch clamp pipettes, we use Sutter boar silk at capillaries that are clean and dust free, and we use the Sutter P nine seven flaming ground micro electrode puller. Sutter's Pipette Cookbook is an excellent reference for developing suitable pulling programs.

To begin, lift the dust cover on the puller. Then take one glass capillary tube from the box. Make sure to replace the lid to minimize dust accumulation.

Next, with one hand, slide the capillary into the groove on one side of the filament, slightly tighten the clamp. At this point, the clamp should be tight enough to prevent the tube from falling, but loose enough that you can slide it into the groove. Now, release the spring metal stop on the same side.

Then with your thumb and forefinger grip the metal tabs that are attached to the bottom of both carriers. Release the other spring metal stop. Pull the carriers together as far as they will go and hold in place with one hand.

Next, slide the tube through the filament housing into the groove of the carrier on the other side. Avoid pushing the glass into the heating filament. Tighten the clamps on both sides.

They should be tight enough to hold the carriers in place, but not so tight that the glass is crushed. Now, close the dust cover and press the green button. Marked pole pipettes for patch clamp recordings are formed using a five step polling program with descending heat and velocity at each step and a small pole on the final step.

This program uses heat and gravity. The procedure for making sharp electrodes is very similar, but in this case, we use the thin glass capillary from Warner Instruments that has an outer diameter of one millimeter and an inner diameter of 0.78 millimeters. We also apply a pulling program that uses a motor in addition to heat and gravity when the pulling programs are finished.

Lift the dust cover, loosen both clamps and remove the finished pipette. Then put the pipettes in a working box. The box can be made by gluing a strip of foam to the rack of a plastic pipette tip box, and then cutting six to eight slits in the foam.

Insert the pipettes into the slits. Once the box is full of pulled pipettes, proceed to the fire polishing step to fire polish the pipettes. Set up a micro forge rig with a platinum heating filament controlled by a foot pedal before starting the fire polishing.

First coat the pipette with dental wax to decrease capacitance and improve noise characteristics. To do this, keep a small supply of molten wax nearby. Apply air pressure on the back of the pipette to keep wax from entering the tip.

Then dip the tip briefly into the liquid wax. And remove now. Place the pipette in the polishing apparatus and bring the tip approximately 50 microns from the filament.

Keep in mind that the filament will expand when heated. Apply a brief heat pulse of one or two seconds to remove the wax from the tip of the pipette and smooth the glass. Examine the pipette tips under a microscope to determine opening diameter and smoothness.

For standard patch clamp recordings, tip openings should be one to three microns in diameter, discard, rough, uneven, or irregular tips. Place the finished pipette in a working box, making sure to close the lid to protect the pipettes from dust repeat for 10 successful pipettes. I've just shown you how to make patch clamp pipettes and sharp electros using a programmable puller.

The same procedure can be used to make a variety of glass tools, including injection needles for optimal patch clamp results. It's important to remember to pull new dust-free pipettes on the same day of the experiment and to polish them within an hour of the experiment. Also verify by inspection that the pipettes are smooth and polished.

Be sure to watch our other video, which demonstrates how to perform patch clamp recording of ion channels expressed in opus oocytes. Okay, that's it. Now you're a pipette expert and good luck with your experiments.