Name: pressure-polishing pipettes for improved patch-clamp recording
Uploaded: 2008-10-22T00:00:00
Description: Watch this Scientific Journal Video about Pressure-polishing Pipettes for Improved Patch-clamp Recording at JoVE.com

<p class="jove_content">The pressure-polishing technique was developed in collaboration with S. R. Lockery.</p>

<p class="jove_step">Pulling pipettes</p>
<p class="jove_content">Using a filament puller such as a Sutter P-97 Flaming/Brown Microelectrode Puller, pull a set of approximately 10-20 pipettes.</p>
<ol>
<li>Select your glass. We use borosilicate (BF150-86-10, OD = 1.5 mm, ID = 0.86 mm). Store the glass carefully so it remains clean and dust free.</li>
<li>Design a pulling program. The primary goal is a tip opening approximately the desired size, which will depend on application. Sutter's <em>P-97 Pipette Cookbook </em><sup>2</sup> is an excellen...

<ol>
	<li>Goodman, M. B., Lockery, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pressure+polishing:+a+method+for+re-shaping+patch+pipettes+during+fire+polishing. .">Pressure polishing: a method for re-shaping patch pipettes during fire polishing. .</a> <em style='font-style: italic'>J Neurosci Methods</em>. <b>100</b>, 13-15 (2000).</li></ol>

<p class="jove_content">We originally developed this method to make pipettes useful for patch-clamp recording from 2-3 &micro;m neurons in <em>C. elegans</em> <sup>1</sup>. It is in routine use in the Goodman laboratory. We pressure-polish pipettes not only for recording from tiny <em>C. elegans</em> neurons, but also for obtaining macropatch and single-channel recordings of ion channels expressed in Xenopus oocytes. For a given tip-opening diameter, the blunt shape produces a lower-resistance pipette. Lower pipette resistance leads to lower se...

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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<tr>
<td>Glass Capillaries</td>
<td>Consumable</td>
<td>Sutter Instruments</td>
<td>BF150-85-10 (or similar)</td>
<td>The method works with any type of capillary except those made from quartz.  However, we recommend using capillaries with a filament.</td>
</tr>
<tr>
<td>Inverted Microscope</td>
<td>Microscope</td>
<td>Leica</td>
<td>DM IL (or similar)</td>
<td>Any simple inverted microscope will do.  However, it is essential to have sufficient magnification to see pipettes during polishing.  We use a 100x metallurgical lens with a long working distance for this purpose.</td>
</tr>
<tr>
<td>Microforge Kit</td>
<td>Tool</td>
<td>ALA Science</td>
<td>CPM-2</td>
<td>Supplies polishing coil, controller and foot pedal.</td>
</tr>
<tr>
<td>Air pressure source</td>
<td>Tool</td>
<td>ALA Science</td>
<td>PR-60</td>
<td>Accessory for providing high pressure, includes a suitable valve.  Alternatively, compressed air from the building can be used together with a filter/regulator (set to 40 PSI) and a 4-way valve (see below)</td>
</tr>
<tr>
<td>4-way valve</td>
<td>Tool</td>
<td>Cole-Parmer</td>
<td>A-98150-01</td>
<td>Fittings will be needed to couple valve to tubing</td>
</tr>
<tr>
<td>Pipette Holder with Luer Connector</td>
<td>Tool</td>
<td>World Precision Instruments</td>
<td>MPH-6S20</td>
<td>Use a male luer-to-barb fitting to connect pipette holder to high-pressure tubing</td>
</tr>
<tr>
<td>Tygon&#174; tubing</td>
<td>Tool</td>
<td>Cole-Parmer</td>
<td>06408-62</td>
<td>Use this tubing to connect pipette holder to 4-way valve
</td>
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pressure-polishing pipettes for improved patch-clamp recording

<p class="jove_content">Pressure-polishing is a method for shaping glass pipettes for patch-clamp recording.  We first developed this method for fabricating pipettes suitable for recording from small (&lt;3  m) neuronal cell bodies.  The basic principal is similar to glass-blowing and combines air pressure and heat to modify the shape of patch pipettes prepared by a conventional micropipette puller.  It can be applied to so-called soft (soda lime) and hard (borosilicate) glasses. Generally speaking, pressure polishing can reduce pipette resistance by 25% without decreasing the diameter of the tip opening (Goodman and Lockery, 2000).  It can be applied to virtually any type of glass and requires only the addition of a high-pressure valve and fitting to a microforge.  This technique is essential for recording from ultrasmall cells (&lt;5  m) and can also improve single-channel recording by minimizing pipette resistance.  The blunt shape is also useful for perforated-patch clamp recording since this tip shape results in a larger membrane bleb available for perforation.</p>

Watch this Scientific Journal Video about Pressure-polishing Pipettes for Improved Patch-clamp Recording at JoVE.com

Pressure-polishing Pipettes for Improved Patch-clamp Recording

<p class="jove_content">This is a guide to modifying the shape of glass micropipettes.  Specifically, by using heat and air pressure the taper is widened without increasing the tip opening, leading to lower pipette resistance.  This is critical to obtain low noise recordings of small cells but is useful in many applications.</p>

Pressure-polishing is a method for shaping glass pipettes for patch-clamp recording.  We first developed this method for fabricating pipettes suitable for ...

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