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Making Patch-pipettes and Sharp Electrodes with a Programmable Puller

1, 2, 2

1Department of Molecular and Cellular Physiology, Stanford University, 2Department of Molecular and Cellular Physiology, Stanford University School of Medicine

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Cite this Article: Making Patch-pipettes and Sharp Electrodes with a Programmable Puller

L. Brown, A., E. Johnson, B., B. Goodman, M. Making Patch-pipettes and Sharp Electrodes with a Programmable Puller. J. Vis. Exp. (20), e939, doi:10.3791/939 (2008).

Abstract: 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 <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.

Protocol: Making Patch-pipettes and Sharp Electrodes with a Programmable Puller

Pulling pipettes

Using a microelectrode puller such as the Sutter P-97 Flaming/Brown, pull a set of approximately 10-20 pipettes.

  1. 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.
  2. 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.
  3. 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.

Fire polishing pipettes

  1. 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.
  2. Optional: Coat the pipette with an insulator to decrease capacitance and improve noise characteristics. We use dental wax. Sylgard 184™ (poly-dimethyl siloxane elastomer also known as PDMS) is also an option.
  3. 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.
  4. 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.
  5. Recommended: Follow the separate protocol for "pressure polishing" to change the shape of the pipette for optimal resistance and tip diameter.
  6. A brief heat pulse (1-2 seconds) is sufficient to remove wax from the tip of the pipette and smooth the glass.
  7. Place the finished pipette in a closed box to protect from dust, repeat for 10 successful pipettes.

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Discussion: Making Patch-pipettes and Sharp Electrodes with a Programmable Puller

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.  With attention and care, the filament on your puller will last for one year or more.  Good luck with your experiments.

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Disclosures: Making Patch-pipettes and Sharp Electrodes with a Programmable Puller

Acknowledgements: Making Patch-pipettes and Sharp Electrodes with a Programmable Puller

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.

Materials: Making Patch-pipettes and Sharp Electrodes with a Programmable Puller

Name Type Company Catalog Number Comments
Micropipette Puller Instrument Sutter Instrument Co. P-97 Or similar instrument (e.g. Sutter P-87 or P-2000)
Glass Capillaries Reagent Sutter Instrument Co. BF150-86-10 Or, similar capillary glass. To make filling the pipette easier, use a capillary with a glass filament.

References: Making Patch-pipettes and Sharp Electrodes with a Programmable Puller

1. Sutter Instrument, P-97 Pipette Cookbook, 2008 (rev. D) http://www.sutter.com/contact/faqs/pipette_cookbook.pdf

Ask the Author: Making Patch-pipettes and Sharp Electrodes with a Programmable Puller

3 Comments

This video is just misleading any new students that pulling patch pipetts are that simple. In reality, without knowing how to program the puller, how to choose right filaments-box or other types, how to instal and allign the filament..............pipette pulling is really incomplete story.

To all students who will watch this video, I encourage to start with cook book and choose right cappilary tubes for your application. Don't take the words for granted that 1-3 micron tip is good for patching. Also, you don't have to always fire polish the capillary. Readymade fire polish capillary are available as well.

 

1

Reply

Posted by: Ruchi ParekhOctober 31, 2008, 1:19 AM

Dear Austin L. Brown, Brandon E. Johnson, Miriam B. Goodman, Department of Molecular and Cellular Physiology, Stanford University

Thanks for demonstrating the art of pulling and making patch type pipettes.

Five things to note:

1) When patching cultured cells, it is best to use BF150-86-10 as you demonstrate in the video. You can use a one line program that loops 4-5 times and it is best to use a midpoint velocity (instructions found on page 26 in the cookbook) to have a stable and reliable program. If for some reason the tip size or resistance is not exactly as you need, you can then write the program out into a 4 or 5 line program and adjust the heat or velocity on the last line to better control the tip morphology. I personally would not create a program with gradually descending heats and velocities as this seems a bit labor intensive and maybe confusing. It could also introduce some variability. But, if it works, stick with it. There are many roads leading to the same point (so to speak), and as long as your program is stable, you will have a high yield of usable pipettes.

2) When pulling patch pipettes for slice or whole tissue, it is best to use thin walled glass BF150-110-10 and a one line program that loops two times instead of 4-5. This will provide a slightly longer  taper and a more gradual approach to the tip. This more gradual taper is best for inserting the pipette through multiple cell/tissue layers as it reduces damage to the tissue.

3) Always run a ramp test when writing a new program or using a program/puller you are not familiar with. When using a box filament (which is best to generate short tapers and high cone angles), it is best to use the ramp value for the heat setting. When using a trough filament (which generates longer tapers) it is best to use ramp+10 or ramp+15 for your heat setting.

4) The NEW P-1000 pipette puller (which is being shown at the Neuroscience meeting in December 2008 in DC) has a Cookbook feature on the touch screen menu where you can search for and install a cookbook program. Starting programs can be found by designating the filament type, glass size, and application. It will then produce a program with which to get started....so there is less guess work! It also has a "Safe Heat Mode" which will reduce the incidence of burning out the filament.

5) When pulling sharp pipettes, you can go to the General Look Up Tables at the end of the Cookbook. Here you will find Type, A, B, C, D, and E programs. I recommend starting with a Type A for PAtch, and a Type B or C for sharp electrodes where the resistances are >20 Megohms.

If anyone out there need more help, feel free to call Sutter 415-883-0128 and ask for me (Adair) and I will be happy to help with any additional concerns.

Sincerely, Adair

2

Reply

Posted by: adairOctober 31, 2008, 2:36 PM

To examine the obtained pipette (5-10micrometer radii) under a microscope, is the microscope special properties (stereo, invert, ....)? Could you infrom me about it, please? Thank you.

4

Reply

Posted by: Haluk B.February 6, 2009, 4:20 PM

We use a Leica DM IL inverted microscope with a pl fluotar 100x objective.  The key is to have a high magnification, long-working distance objective.

4.1

Reply

Posted by: Brandon JohnsonFebruary 6, 2009, 5:54 PM

Thank you very much Mr. B.Johnson.

Sincerely....

4.1.1

Reply

Posted by: Haluk B.February 7, 2009, 9:53 PM

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