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| | </font> | | </font> |
| | </p> | | </p> |
| | + | <br><br> |
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| - | <p text-aligne:left style="margin-left:60px; margin-right:50px"><font size="3" color="#F0F8FF" face="Arial regular">
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| - | <br>
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| - | <B>Chemicals & consumables<br></B>
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| - | <div align="left" style="margin-left:60px; margin-right:50px">
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| - | <ul>
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| - | <li class=list1>- SDS</li>
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| - | <li class=list1>- Rotiphorese® (30%)</li>
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| - | <li class=list1>- Tris HCl</li>
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| - | <li class=list1>- Glycine</li>
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| - | <li class=list1>- TEMED</li>
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| - | <li class=list1>- APS</li>
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| - | <li class=list1>- Aqua dest.</li>
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| - | <li class=list1>- Isopropyle alcohol</li>
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| - | <li class=list1>- Glycerine</li>
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| - | <li class=list1>- Beta-Mercaptoethanol</li>
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| - | <li class=list1>- Bromphenolblue</li>
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| - | <li class=list1>- Coomassie brilliant blue G250</li>
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| - | <li class=list1>- Coomassie brilliant blue R250</li>
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| - | <li class=list1>- Methanol</li>
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| - | <li class=list1>- Acetate (99%)</li>
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| - | </ul>
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| - | </div>
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| - | </font>
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| - | </p>
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| - |
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| - | <p text-aligne:left style="margin-left:60px; margin-right:50px"><font size="3" color="#F0F8FF" face="Arial regular">
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| - | <br>
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| - | <B>Buffers & gels<br></B>
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| - | <div align="left" style="margin-left:60px; margin-right:50px">
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| - | <ul>
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| - | <li class=list1>- Separation buffer (0.5 M Tris, 0.4% SDS, pH = 8.8)</li>
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| - | <li class=list1>- Stacking buffer (0.5 M Tris, 0.4% SDS, pH = 6.6)</li>
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| - | <li class=list1>- Running buffer (0.25 M Tris, 2 M glycine, 1% SDS , pH = 8.3)</li>
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| - | <li class=list1>- Separation gel 12.5% (5 ml Separation buffer, 6.25 ml Rotiphorese®, 3.75 ml Aqua dest., 30 µl TEMED, 30 µl APS (40%))</li>
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| - | <li class=list1>- Stacking gel 4% (3 ml Separation buffer, 1.33 ml Rotiphorese®, 5.67 ml Aqua dest.,20 µl TEMED, 20 µl APS (40%)</li>
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| - | <li class=list1>- 3x Sample buffer (65 mM Tris, 4% SDS, 20% glycerine, 10% beta-Mercaptoethanol, 1 tip of bromphenolblue, pH = 6.75)</li>
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| - | <li class=list1>- Staining buffer (0.5 g Coomassie brilliant blue G250 & R250 each, 100 ml methanol, 100 ml Aqua dest., 20 ml acetate)</li>
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| - | <li class=list1>- Destaining buffer (400 ml Aqua dest., 100 ml methanol, 30 ml acetate)</li>
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| - | </ul>
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| - | </div>
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| - | </font>
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| - | </p>
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| - | <br>
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| | <p text-aligne:left style="margin-left:50px; margin-right:50px"><font size="4.5" color="#F0F8FF" face="Arial regular"> | | <p text-aligne:left style="margin-left:50px; margin-right:50px"><font size="4.5" color="#F0F8FF" face="Arial regular"> |
| | <B> Procedure<br></B></font></p> | | <B> Procedure<br></B></font></p> |
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| | <br> | | <br> |
| | <B><li class=list1>Load & Run</li></B> | | <B><li class=list1>Load & Run</li></B> |
| - | <li>prepare the separating gel and fill it into the chamber </li> | + | <li>Inoculation of 50 mL DYT medium in the 100 mL flask with <i>E. coli</i> BL21 DE3 containing <a href=http://lucerna-chem.ch/shop/558271> pPR-IBA2 </a> |
| - | <li>pour 1 mL isopropyle alcohol on the top of the gel to destroy air bubbles and prevent dehydration </li>
| + | plasmid with the sequence for the respective part to be expressed.</li> |
| - | <li>discard the isoprpyl alcohol and pour the prepared stacking gel</li>
| + | <li>Incubation at 180 repulsion per minute (rpm) at 30°C to an OD600= 4</li> |
| - | <li>stick in the comb</li>
| + | <li>Transferation of the starter culture into 1 L DYT medium in a 3 L flask resulting in an OD600= 0.2</li> |
| - | <li>if not used, store the gel in wet cloth (to prevent dehydration) at 4°C</li>
| + | <li>Incubation to an OD600= 0.6 at 180 rpm and 30°C.</li> |
| - | <li>if used, remove comb when the gel is solid and place it into SDS PAGE chamber</li>
| + | <li>Incubation for 15 minutes on ice.</li> |
| - | <li>fill chamber with running buffer</li>
| + | <li>Induction of the proteinexpression with 0.8 mL of IPTG (stock conentration 1mM).</li> |
| - | <li>after heating the samples with 3x sample buffer at 70°C for 15 min, apply 20 µl to each pocket</li>
| + | <li>Incubation of the cell suspension over night at 180 rpm at 16°C.</li> |
| - | <li>load most outer pocket with a commercial protein marker</li> | + | |
| - | <li>start the PAGE by applying 20 mA / gel at stacking</li>
| + | |
| - | <li>apply 40 mA / gel at separation </li>
| + | |
| - | </ol>
| + | |
| - | <br>
| + | |
| - | <ol>
| + | |
| - | <B><li class=list1>Staining & washing of gel</li></B>
| + | |
| - | <li>disconnect glas plates containing the already run gel</li>
| + | |
| - | <li>cut off the stacking gel</li> | + | |
| - | <li>put the separation gel into the staining buffer and let it shake at room temperature for at least one hour</li> | + | |
| - | <li>put the stained separation gel into the destaining buffer and let it shake for 5 minutes</li> | + | |
| - | <li>repeat the previous step at least twice again with fresh destaining buffer each</li> | + | |
| | </ol> | | </ol> |
| - | </div>
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| - | </font></p>
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| - | <a href=http://lucerna-chem.ch/shop/558271> pPR-IBA2 </a>
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