Team:TU Darmstadt/protocols/Protein Expression

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<img alt="Problem" src="/wiki/images/6/66/Darmstadt_green_Problem.jpg" width="100" height="30"></a>
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<a href="https://2013.igem.org/Team:TU_Darmstadt/solution">
 
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<img alt="solution" src="/wiki/images/8/87/Darmstadt_green_Solution.jpg" width="100" height="30"></a>
 
<a href="https://2013.igem.org/Team:TU_Darmstadt/result">
<a href="https://2013.igem.org/Team:TU_Darmstadt/result">
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<br>
<br><br><br><br>
<br><br><br><br>
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<center>
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<a href="https://2013.igem.org/Team:TU_Darmstadt/labbook">
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<font size="8" color="#F0F8FF" face="Arial regular">Lab book |</font>
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</a>
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<a href="https://2013.igem.org/Team:TU_Darmstadt/materials">
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<font size="8" color="#F0F8FF" face="Arial regular">Materials |</font>
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</a>
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<a href="https://2013.igem.org/Team:TU_Darmstadt/protocols">
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<font size="8" color="#F0F8FF" face="Arial regular">Protocols</font>
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</a>
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</center>
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<br>
<h2><font size="6" color="#F0F8FF" face="Arial regular">Protein Expression</font></h2>
<h2><font size="6" color="#F0F8FF" face="Arial regular">Protein Expression</font></h2>
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<body>
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<p text-aligne:left style="margin-left:50px; margin-right:50px">
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<B> Materials<br></B></font></p>
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<p text-aligne:left style="margin-left:60px; margin-right:50px"><font size="3" color="#F0F8FF" face="Arial regular">
<p text-aligne:left style="margin-left:60px; margin-right:50px"><font size="3" color="#F0F8FF" face="Arial regular">
<br>
<br>
-
<B>Equipment & chemicals<br></B>
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<B>Equipment<br></B>
<div align="left" style="margin-left:60px; margin-right:50px">
<div align="left" style="margin-left:60px; margin-right:50px">
<ul>
<ul>
<li class=list1>- Incubation shaker</li>
<li class=list1>- Incubation shaker</li>
-
<li class=list1>- <i>E. coli</i> BL21 DE3</li>
 
-
<li class=list1>- DYT media</li>
 
-
<li class=list1>- IPTG</li>
 
-
<li class=list1>- 100 ml and 3 l flasks</li>
 
<li class=list1>- Photometer</li>
<li class=list1>- Photometer</li>
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<li class=list1>- ice</li>
 
</ul>
</ul>
</div>
</div>
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<div align="left" style="margin-left:60px; margin-right:50px">
<ul>
<ul>
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<li class=list1>- SDS</li>
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<li class=list1>- E. coli BL21 DE3</li>
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<li class=list1>- Rotiphorese® (30%)</li>
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<li class=list1>- <a href="https://2013.igem.org/Team:TU_Darmstadt/materials/Media"><font size="3" color="#F0F8FF" face="Arial regular"><b>DYT Medium</b></font></a></li>
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<li class=list1>- Tris HCl</li>
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<li class=list1>- IPTG</li>
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<li class=list1>- Glycine</li>
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<li class=list1>- 100 ml and 3 l flasks</li>
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<li class=list1>- TEMED</li>
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<li class=list1>- ice</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>
</ul>
</div>
</div>
</font>
</font>
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<p text-aligne:left style="margin-left:60px; margin-right:50px"><font size="3" color="#F0F8FF" face="Arial regular">
 
-
<br>
 
-
<B>Buffers & gels<br></B>
 
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<div align="left" style="margin-left:60px; margin-right:50px">
 
-
<ul>
 
-
<li class=list1>- Separation buffer (0.5 M Tris, 0.4% SDS, pH = 8.8)</li>
 
-
<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>
 
-
<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>
 
-
<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>
 
-
<li class=list1>- Destaining buffer (400 ml Aqua dest., 100 ml methanol, 30 ml acetate)</li>
 
-
</ul>
 
-
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-
</font>
 
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-
<br>
 
<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>
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<B> Procedure<br></B></font></p><br>
<p text-aligne:left style="margin-left:60px; margin-right:60px"><font size="3" color="#F0F8FF" face="Arial regular">
<p text-aligne:left style="margin-left:60px; margin-right:60px"><font size="3" color="#F0F8FF" face="Arial regular">
-
 
-
 
<div align="left" style="margin-left:30px; margin-right:50px">
<div align="left" style="margin-left:30px; margin-right:50px">
<ol>
<ol>
-
<br>
+
<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><font size="3" color="#F0F8FF" face="Arial regular"><b> pPR-IBA2 </b></font></a>
-
<B><li class=list1>Load & Run</li></B>
+
plasmid with the sequence for the respective part to be expressed.</li>
-
<li>prepare the separating gel and fill it into the chamber </li>
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<li>Incubation at 180 repulsion per minute (rpm) at 30°C to an OD600= 4</li>
-
<li>pour 1 mL isopropyle alcohol on the top of the gel to destroy air bubbles and prevent dehydration </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>discard the isoprpyl alcohol and pour the prepared stacking gel</li>
+
<li>Incubation to an OD600= 0.6 at 180 rpm and 30°C.</li>
-
<li>stick in the comb</li>
+
<li>Incubation for 15 minutes on ice.</li>
-
<li>if not used, store the gel in wet cloth (to prevent dehydration) at 4°C</li>
+
<li>Induction of the proteinexpression with 20 mL of IPTG (stock conentration 1M).</li>
-
<li>if used, remove comb when the gel is solid and place it into SDS PAGE chamber</li>
+
<li>Incubation of the cell suspension over night at 180 rpm at 30°C.</li>
-
<li>fill chamber with running buffer</li>
+
-
<li>after heating the samples with 3x sample buffer at 70°C for 15 min, apply 20 µl to each pocket</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>
</div>
</font></p>
</font></p>
-
<a href=http://lucerna-chem.ch/shop/558271> pPR-IBA2 </a>
 

Latest revision as of 01:59, 5 October 2013








Lab book | Materials | Protocols

Protein Expression

Materials


Equipment

  • - Incubation shaker
  • - Photometer


Chemicals & consumables

  • - E. coli BL21 DE3
  • - DYT Medium
  • - IPTG
  • - 100 ml and 3 l flasks
  • - ice


Procedure


  1. Inoculation of 50 mL DYT medium in the 100 mL flask with E. coli BL21 DE3 containing pPR-IBA2 plasmid with the sequence for the respective part to be expressed.
  2. Incubation at 180 repulsion per minute (rpm) at 30°C to an OD600= 4
  3. Transferation of the starter culture into 1 L DYT medium in a 3 L flask resulting in an OD600= 0.2
  4. Incubation to an OD600= 0.6 at 180 rpm and 30°C.
  5. Incubation for 15 minutes on ice.
  6. Induction of the proteinexpression with 20 mL of IPTG (stock conentration 1M).
  7. Incubation of the cell suspension over night at 180 rpm at 30°C.

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