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[GUIDELINES]
<div><strong>GEL PREPARATIONS</strong></div>
<p><strong><br />Sample Recipe for Resolving Gel:<br /></strong></p>
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<tbody>
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<td>12.5% acrylamide (40.0 mL volume)</td>
</tr>
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<td>16.7 mL 30% acrylamide solution</td>
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<td>10.0 mL 4X resolving gel buffer</td>
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<td>12.4 mL d-H<sub>2</sub>O</td>
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<td>0.4 mL 10% SDS</td>
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<td>20.0 µL TEMED</td>
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<td>0.5 mL 10% Ammonium persulfate (APS) - make fresh</td>
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</tbody>
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<p><br />For gradient gels, a 4 M to 8 M urea gradient is included in the gel to stabilize the acrylamide gradient and to aid in maximal denaturation of the polypeptides.<br /><br /> Remember that when making volume calculations that 1.0 gm of urea is equivalent to 0.76 mL of d-H<sub>2</sub>O. To achieve an acrylamide concentration greater than about 12% in the 8 M urea, the concentration of the resolving gel buffer or the acrylamide stock solution needs to be increased. If heat is used to get the 8 M urea into solution remember to let the solution cool to room temperature before pouring the gradient.<br /><br /> The amount of TEMED and ammonium persulfate are reduced when pouring a gradient gel compared to a straight percentage gel to allow ample time for pouring the gradient before polymerization of the gel.<br /><br /><strong>Sample Recipe for Gradient Gel:<br /></strong><br />(for 250 mm gel, 1.5 mm spacers)</p>
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<td style="height: 20px; width: 116px;">  </td>
<td style="height: 20px; width: 98px;"><strong>7.5% Acrylamide</strong><br /><strong>4 M Urea</strong></td>
<td style="height: 20px; width: 92px;"><strong>15.0% Acrylamide</strong><br /><strong>8 M Urea</strong></td>
</tr>
<tr style="height: 20px;">
<td style="height: 20px; width: 116px;">30.0% acrylamide</td>
<td style="height: 20px; width: 98px;">6.3 mL </td>
<td style="height: 20px; width: 92px;">12.5 mL</td>
</tr>
<tr style="height: 20px;">
<td style="height: 20px; width: 116px;">4X resolving buffer</td>
<td style="height: 20px; width: 98px;">6.25 mL</td>
<td style="height: 20px; width: 92px;">---</td>
</tr>
<tr style="height: 20px;">
<td style="height: 20px; width: 116px;">10X resolving buffer</td>
<td style="height: 20px; width: 98px;">--- </td>
<td style="height: 20px; width: 92px;">2.5 mL</td>
</tr>
<tr style="height: 20px;">
<td style="height: 20px; width: 116px;">Urea </td>
<td style="height: 20px; width: 98px;">6.0 gm </td>
<td style="height: 20px; width: 92px;">12.0 gm</td>
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<td style="height: 20px; width: 116px;">10% SDS </td>
<td style="height: 20px; width: 98px;">0.25 mL </td>
<td style="height: 20px; width: 92px;">0.25 mL</td>
</tr>
<tr style="height: 24px;">
<td style="height: 24px; width: 116px;">d-H<sub>2</sub>O </td>
<td style="height: 24px; width: 98px;">7.5 mL </td>
<td style="height: 24px; width: 92px;">0.55 mL</td>
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<tr style="height: 20px;">
<td style="height: 20px; width: 116px;">TEMED</td>
<td style="height: 20px; width: 98px;">7.5 µL </td>
<td style="height: 20px; width: 92px;">7.5 µL </td>
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<tr style="height: 20px;">
<td style="height: 20px; width: 116px;">10% APS</td>
<td style="height: 20px; width: 98px;">87.0 µL </td>
<td style="height: 20px; width: 92px;">87.0 µL</td>
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</tbody>
</table>
<p> </p>
<p>Mix all of the ingredients together except the ammonium persulfate.  Don’t add the ammonium persulfate until just before pouring the gel.</p>
<p> </p>
<p>Gently overlay the gel with d-H<sub>2</sub>O (or 1X resolving gel buffer) and allow the gel to polymerize.  The line between the gel and the water will first fade away and then become very sharp when the gel has polymerized.</p>
<p> </p>
<p><strong>Sample Recipe for Stacking Gel</strong></p>
<p> </p>
<p>(for 250 mm gel, 1.5 mm spacers)</p>
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<td style="width: 88px; height: 48px;"> </td>
<td style="width: 105px; height: 48px;">
<p><strong>4.5% Acrylamide</strong></p>
<p><strong>No Urea</strong></p>
</td>
<td style="width: 104px; height: 48px;">
<p><strong>4.5 % Acrylamide</strong></p>
<p><strong>4 M Urea</strong></p>
</td>
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<tr style="height: 20px;">
<td style="width: 88px; height: 20px;">Urea</td>
<td style="width: 105px; height: 20px;">---</td>
<td style="width: 104px; height: 20px;">2.4 gm</td>
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<tr style="height: 40px;">
<td style="width: 88px; height: 40px;">30.0% acrylamide</td>
<td style="width: 105px; height: 40px;">1.5 mL</td>
<td style="width: 104px; height: 40px;">1.5 mL</td>
</tr>
<tr style="height: 40px;">
<td style="width: 88px; height: 40px;">4X stacking gel buffer</td>
<td style="width: 105px; height: 40px;">2.5 mL</td>
<td style="width: 104px; height: 40px;">2.5 mL</td>
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<td style="width: 88px; height: 20px;">10% SDS</td>
<td style="width: 105px; height: 20px;">0.1 mL</td>
<td style="width: 104px; height: 20px;">0.1 mL</td>
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<tr style="height: 24px;">
<td style="width: 88px; height: 24px;">d-H<sub>2</sub>O</td>
<td style="width: 105px; height: 24px;">5.8 mL</td>
<td style="width: 104px; height: 24px;">3.9 mL</td>
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<tr style="height: 20px;">
<td style="width: 88px; height: 20px;">TEMED</td>
<td style="width: 105px; height: 20px;">5.0 µL</td>
<td style="width: 104px; height: 20px;">5.0 µL</td>
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<tr style="height: 20px;">
<td style="width: 88px; height: 20px;">10% APS</td>
<td style="width: 105px; height: 20px;">130.0 µL</td>
<td style="width: 104px; height: 20px;">130.0 µL</td>
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</tbody>
</table>
<p> </p>
<p>Pour the stacking gel immediately after adding the ammonium persulfate.  Insert the gel comb to form the wells.  Allow the stacking gel to polymerize for 60 min before use.</p>
<p> </p>
<p>Boil the samples in the cracking buffer 5-10 min before loading onto the gel.</p>
<p> </p>
<p>Electrophorese the gel at 100 volts (constant) or 10 milliamps (constant) until the dye front just runs off the gel.</p>
<p> </p>
<p><br /><strong>MINIGELS<br /><br />Resolving Gel Recipes:<br /><br /></strong>5.0 mL gel </p>
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<td> </td>
<td><strong>9.0% </strong></td>
<td><strong>10.0%</strong></td>
<td><strong>12.5%</strong></td>
<td><strong>15.0%</strong></td>
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<td>30.0% acrylamide</td>
<td>1.50 mL</td>
<td>1.67 mL</td>
<td>2.10 mL</td>
<td>2.50 mL</td>
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<td>4X resolving gel buffer</td>
<td>1.25 mL</td>
<td>1.25 mL</td>
<td>1.25 mL</td>
<td>1.25 mL</td>
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<td>20% SDS</td>
<td>25.0 µL</td>
<td>25.0 µL</td>
<td>25.0 µL</td>
<td>25.0 µL</td>
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<td>d-H2O </td>
<td>2.25 mL</td>
<td>2.08 mL</td>
<td>1.65 mL</td>
<td>1.25 mL</td>
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<td>10% APS</td>
<td>62.5 µL</td>
<td>62.5 µL</td>
<td>62.5 µL</td>
<td>62.5 µL</td>
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<td>TEMED </td>
<td>2.5 µL</td>
<td>2.5 µL</td>
<td>2.5 µL</td>
<td>2.5 µL</td>
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</table>
<p><br /><strong>Stacking Gel Recipes:</strong><br /><br /> 2.5 mL gel</p>
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<td> </td>
<td><strong>3.0%</strong></td>
<td><strong>3.5%</strong></td>
<td><strong>4.5%</strong></td>
<td><strong>5.0%</strong></td>
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<td>30.0% acrylamide</td>
<td>0.25 mL</td>
<td>0.292 mL</td>
<td>0.375 mL</td>
<td>0.417 mL</td>
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<td>4X stacking gel buffer</td>
<td>0.625 mL</td>
<td>0.625 mL</td>
<td>0.625 mL</td>
<td>0.625 mL</td>
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<td>20% SDS</td>
<td>12.5 µL</td>
<td>12.5 µL</td>
<td>12.5 µL</td>
<td>12.5 µL</td>
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<td>d-H<sub>2</sub>O</td>
<td>1.625 mL</td>
<td>1.583 mL</td>
<td>1.45 mL</td>
<td>1.458 mL</td>
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<td>10% APS</td>
<td>32.5 µL</td>
<td>32.5 µL</td>
<td>32.5 µL</td>
<td>32.5 µL</td>
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<td>TEMED  </td>
<td>1.25 µL</td>
<td>1.25 µL</td>
<td>1.25 µL</td>
<td>1.25 µL</td>
</tr>
</tbody>
</table>
<p> </p>
<p> </p>
<p><strong>COOMASSIE STAINING OF THE GELS </strong></p>
<p> </p>
<p>Stain the gels with coomassie blue stain according to the following recipe:</p>
<p>                           1.0 gm Coomassie Brilliant Blue R</p>
<p>                           455 mL methanol</p>
<p>                           455 mL d-H<sub>2</sub>O</p>
<p>                           90 mL glacial acetic acid</p>
<p> </p>
<p>Gels are destained with the following:</p>
<p>                           700 mL methanol</p>
<p>                           200 mL glacial acetic acid</p>
<p>                           2600 mL d-H<sub>2</sub>O</p>
<p> </p>
<p>Stain and destain the gels at 65<sup>0</sup>C.  Small foam rubber pieces added during the destaining process absorb some of the coomassie with the result that less destain is needed and the destain need not be changed as often.                                                                                                     </p>
<p> </p>
<p> </p>
<p> </p>

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