Fabrication of Patch Pipets

J.L. Rae1, R.A. Levis2

1 Mayo Foundation, Rochester, Minnesota, 2 Rush Medical College, Chicago, Illinois
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 6.3
DOI:  10.1002/0471142301.ns0603s26
Online Posting Date:  May, 2004
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Abstract

Patch clamping refers to a wide range of electrophysiological measurements, all of which have in common the use of patch pipets and the formation of gigaohm seals. The purpose of this unit is to describe the fabrication of patch pipets. The aspects of the pipet geometry that are important to different applications and the different procedures that have been found to most reliably and simply achieve these results are described. Parameters for glass selection are detailed in the beginning of the unit. Pulling patch and whole‐cell pipets, elastomer coating, fire polishing, pipet filling, and pipet testing in an experimental setup are highlighted. Additional support protocols describe alternative ways to optimize pipet geometry and cleaning the glass before pulling. Considerations for noise and dynamic performance are emphasized as these two requirements for single‐channel and whole‐cell current measurements dictate how the pipets must be fabricated.

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Pulling Single‐Channel and Whole‐Cell Electrodes with an Automated Puller
  • Support Protocol 1: Obtaining Optimal Tip Geometry
  • Support Protocol 2: Calibrating the Puller Filament
  • Support Protocol 3: Cleaning the Glass
  • Support Protocol 4: Noise Considerations for Single‐Channel Patch Pipets
  • Support Protocol 5: Considerations for Whole‐Cell Pipets
  • Basic Protocol 2: Preparing Pipet Tips with Elastomer Coating
  • Support Protocol 6: Considerations for Pipet Coating
  • Basic Protocol 3: Fire Polishing the Pipet
  • Support Protocol 7: Constructing Fire‐Polishing Apparatus Components
  • Basic Protocol 4: Pipet Filling
  • Basic Protocol 5: Mounting and Testing the Pipet Setup
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Pulling Single‐Channel and Whole‐Cell Electrodes with an Automated Puller

  Materials
  • Pipet glass (Garner Glass)
  • Pipet puller (Sutter Instrument)
  • Micropipet storage jar (World Precision Instruments)

Support Protocol 1: Obtaining Optimal Tip Geometry

  Materials
  • Box filament
  • Fine forceps
  • Pipet glass
  • Pipet puller

Support Protocol 2: Calibrating the Puller Filament

  Materials
  • Pipet glass
  • Ethanol or methanol
  • Ultrasonic bath cleaner
  • 100°C oven

Support Protocol 3: Cleaning the Glass

  Materials
  • 1‐ to 2‐mm‐o.d. × 5‐ to 7.6‐cm (2‐ to 3‐in.) glass tubing or rod (World Precision Instruments)
  • Pulled pipet (see protocol 1)
  • Elastomer: RTV615 (General Electric), R‐6101 (Dow Corning), or Sylgard 184 (Dow Corning)
  • Dissecting microsope, preferably modified for dark‐field illumination (Fig. )
  • Heat gun (e.g., Master Model 10008, Newark Electronics)

Support Protocol 4: Noise Considerations for Single‐Channel Patch Pipets

  Materials
  • 100×, long‐working‐distance metallurgical objective with 210‐mm tube length or infinity corrected (e.g., Nikon, Olympus)
  • Fire‐polishing wire: 0.003‐mm platinum‐iridium wire (AM Systems)

Support Protocol 5: Considerations for Whole‐Cell Pipets

  Materials
  • 1‐ml tuberculin and 10‐ml syringes (Becton Dickinson)
  • Fire‐polished pipet (see protocol 9)
  • Suction apparatus
  • 2.0‐mm holder (World Precision Instruments)
  • Needle to fit into the bore of the pipet: e.g., 28‐G Microfil (World Precision Instruments), 1.5‐in. 22‐G Monoject needle, or 1.25‐in. 27‐G Monoject needle

Basic Protocol 2: Preparing Pipet Tips with Elastomer Coating

  Materials
  • Bathing solution appropriate for experiment
  • Pipet
  • Suction line connected to a syringe needle
  • Silver/silver chloride reference electrode
  • Patch clamp apparatus (see unit 6.6)
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Figures

Videos

Literature Cited

Literature Cited
   Benndorff, K. 1995. Low‐noise recording. In Single‐Channel Recording (B. Sakmann and E. Neher, eds.) pp. 129‐145. Plenum, New York.
   Hamill, O.P., Marty, A., Neher, E., Sakmann, B., and Sigworth, F.J. 1981. Improved patch‐clamp techniques for high‐resolution current recording from cells and cell‐free membrane patches. Pflueg. Arch. Eur. J. Physiol. 391:85‐100.
   Levis, R.A. and Rae, J.L. 1992. Constructing a patch‐clamp setup. Methods Enzymol. 207:18‐66.
   Levis, R.A. and Rae, J.L. 1993. The use of quartz patch pipets for low‐noise single‐channel recording. Biophys. J. 65:1666‐1677.
   Levis, R.A. and Rae, J.L. 1995. Technology of patch‐clamp electrodes. In Neuromethods, Vol. 26: Patch‐Clamp Applications and Protocols (A. Boulton, G. Baker and W. Walz, eds.) pp. 1‐36. Humana Press, Totowa, N.J.
   Marty, A. and Neher, E. 1995. Tight‐seal whole‐cell recording. In Single‐Channel Recording (B. Sakmann and E. Neher, eds.) pp. 31‐52. Plenum, New York.
   Neher, E. 1982. Unit conductance studies in biological membranes. In Techniques in Cellular Physiology (P.F. Baker, ed.) pp. 1‐16. Elsevier/North‐Holland, Amsterdam.
   Neher, E. and Sakmann, B. 1976. Single‐channel currents recorded from membrane of denervated frog muscle fibers. Nature 260:799‐802.
   Rae, J.L. and Levis, R.A. 1992a. Glass technology for patch electrodes. Methods Enzymol. 207:66‐92.
   Rae, J.L. and Levis, R.A. 1992b. A method for exceptionally low noise single channel recordings. Pflueg. Arch Eur. J. Physiol. 42:618‐620.
   Rae, J.L. and Levis, R. A. 1994. Patch voltage clamp of lens epithelial cells: Theory and practice. Mol. Physiol. 6:115‐162.
   Sakmann, B. and Neher, E. 1983. Geometric parameters of pipets and membrane patches. In Single‐Channel Recording (B. Sakmann and E. Neher, eds.) pp. 37‐51. Plenum, New York.
   Sigworth, F.J. and Neher, E. 1980. Single Na+ channel currents observed in cultured rat muscle cells. Nature 287:447‐449.
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