Name: Making Patch-pipettes and Sharp Electrodes with a Programmable Puller (Video) | JoVE Journal
Uploaded: 2008-10-08T00:00:00.000+00:00
Duration: 5 min 30 s
Description: Learn to make patch pipettes and sharp electrodes for electrophysiology using a programmable puller. This method demonstrates glass capillary preparation, wax coating, and fire polishing for reliable recordings.

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5 minutes

We thank the following funding agencies and foundations for support: National Institutes of Health, National Science Foundation, American Heart Association, Muscular Dystrophy Association, the Donald B. and Delia E. Baxter Foundation, the Klingenstein Fund and the McKnight Endowment for Neuroscience.

Pulling pipettes
Using a microelectrode puller such as the Sutter P-97 Flaming/Brown, pull a set of approximately 10-20 pipettes.
<ol>
<li>Select your capillary glass. We use borosilicate capillaries (Sutter BF150-110-10, 1.5mm outer diameter, 1.1mm inner diameter, 10cm long). Store the glass carefully so it remains clean and dust free. </li>
<li>Design a pulling program. We use a 5 step program, with descending heat and velocity at each step, and a small pull on the final step. Sutter's pipette cookbook is an excellent reference for developing suitable programs. </li>
<li>Examine the pipette tips under a microscope to determine opening diameter and smoothness. Discard rough, uneven, or irregular tips. A good pulling protocol should ensure these are rare. For standard patch-clamp recording, tip openings should be 1-3 microns in diameter. A steep taper (blunt tip) leads to lower resistance for the same opening diameter. Shape and size can be modified by pressure polishing. </li>
</ol>
Fire polishing pipettes
<ol>
<li>Set up a polishing rig (microforge) with a platinum heating filament controlled by a foot pedal. A useful kit is available from ALA Scientific (CPM-2) or a similar apparatus can be assembled piecemeal.</li>
<li>Optional: Coat the pipette with an insulator to decrease capacitance and improve noise characteristics. We use dental wax. Sylgard 184&trade; (poly-dimethyl siloxane elastomer also known as PDMS) is also an option.</li>
<li>If using wax, keep a small molten supply nearby. With air pressure on the back of the pipette to keep wax from entering the pipette, dip the tip briefly into the liquid wax and remove. With Sylgard, store prepared elastomer in frozen aliquots and paint the pipette tip under a microscope. Heat to cure the elastomer.</li>
<li>Place the pipette (coated or not) in the polishing apparatus and bring the tip ~50 microns from the filament. Keep in mind that the filament will expand when heated.</li>
<li>Recommended: Follow the separate protocol for "pressure polishing" to change the shape of the pipette for optimal resistance and tip diameter.</li>
<li>A brief heat pulse (1-2 seconds) is sufficient to remove wax from the tip of the pipette and smooth the glass.</li>
<li>Place the finished pipette in a closed box to protect from dust, repeat for 10 successful pipettes.</li>
</ol>

<ol><li>Sutter Instrument, P-97 Pipette Cookbook. , Sutter Instrument. 
 
 <a target="_blank" href="http://www.sutter.com/contact/faqs/pipette_cookbook.pdf">http://www.sutter.com/contact/faqs/pipette_cookbook.pdf</a> (2008).</li>
</ol>

The protocol illustrated here is in daily use in electrophysiology laboratories and is also used to make injection needles for cells and animals. With a programmable puller, it is easy to make pipettes for a variety of uses.&nbsp; With attention and care, the filament on your puller will last for one year or more.&nbsp; Good luck with your experiments.

<table><tbody><tr><td>Micropipette Puller</td><td>Instrument</td><td>Sutter Instrument Co.</td><td>P-97</td><td>Or similar instrument (e.g. Sutter P-87 or P-2000)</td></tr><tr><td>Glass Capillaries</td><td>Reagent</td><td>Sutter Instrument Co.</td><td>BF150-86-10</td><td>Or, similar capillary glass. To make filling the pipette easier, use a capillary with a glass filament.</td></tr></tbody></table>

making patch-pipettes and sharp electrodes with a programmable puller

Making Patch-pipettes and Sharp Electrodes with a Programmable Puller

Glass microelectrodes (also called pipettes) have been a workhorse of electrophysiology for decades. Today, such pipettes are made from glass capillaries using a programmable puller. Such instruments heat the capillary using either a metal filament or a laser and draw out the glass using gravity, a motor or both. Pipettes for patch-clamp recording are formed using only heat and gravity, while sharp electrodes for intracellular recording use a combination of heat, gravity, and a motor. The procedure used to make intracellular recording pipettes is similar to that used to make injection needles for a variety of applications, including cRNA injection into Xenopus oocytes. In general, capillary glass &lt;1.2 mm in diameter is used to make pipettes for patch clamp recording, while narrower glass is used for intracellular recording (outer diameter = 1.0 mm). For each tool, the puller is programmed slightly differently. This video shows how to make both kinds of recording pipettes using pre-established puller programs.

This video shows how to use a programmable puller to make patch pipettes and sharp electrodes for electrophysiology. The same procedure can be used to make a variety of glass tools, including injection needles.

Glass microelectrodes (also called pipettes) have been a workhorse of electrophysiology for decades.  Today, such pipettes are made from glass capillaries ...

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.

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Learn to make patch pipettes and sharp electrodes for electrophysiology using a programmable puller. This method demonstrates glass capillary preparation, wax coating, and fire polishing for reliable recordings.

Making Patch-pipettes and Sharp Electrodes with a Programmable Puller

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