Team:TU Darmstadt/protocols/SDS-Page

From 2013.igem.org

(Difference between revisions)
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<li class=list1>- APS</li>
<li class=list1>- APS</li>
<li class=list1>- Aqua dest.</li>
<li class=list1>- Aqua dest.</li>
-
<li class=list1>- Isopropyle alcohol </li>
+
<li class=list1>- Isopropyle alcohol</li>
-
 
+
<li class=list1>- Glycerine</li>
 +
<li class=list1>- Beta-Mercaptoethanol</li>
 +
<li class=list1>- Bromphenolblue</li>
</ul>
</ul>
</div>
</div>
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<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>- Separation buffer (0.5 M Tris, 0.4% g SDS, pH = 8.8)</li>
+
<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% g SDS, pH = 6.6)</li>
+
<li class=list1>- Stacking buffer (0.5 M Tris, 0.4% SDS, pH = 6.6)</li>
-
<li class=list1>- Running buffer (0.25 M Tris, 2 M Glycin, 1% SDS , pH = 8.3)</li>
+
<li class=list1>- Running buffer (0.25 M Tris, 2 M glycine, 1% SDS , pH = 8.3)</li>
<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>- 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>- TEMED</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>
-
<li class=list1>- APS</li>
+
<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>- Aqua dest.</li>
+
 
-
<li class=list1>- Isopropyle alcohol </li>
+
</ul>
</ul>
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<li>discard the isoprpyl alcohol and pour the prepared stacking gel</li>
<li>discard the isoprpyl alcohol and pour the prepared stacking gel</li>
<li>stick in the comb</li>
<li>stick in the comb</li>
-
<li>store the gel in wet cloth (to prevent dehydration) at 4°C</li>
+
<li>if not used, store the gel in wet cloth (to prevent dehydration) at 4°C</li>
</ol>
</ol>
</div>
</div>
</font></p>
</font></p>

Revision as of 19:05, 4 October 2013







Labbook | Matierals | Protocols

Protocols


Equipment

  • - Two glass plates
  • - Gel caster
  • - Comb
  • - VWR Power Source 300V


Chemicals & consumables

  • - SDS
  • - Rotiphorese® (30%)
  • - Tris HCl
  • - Glycine
  • - TEMED
  • - APS
  • - Aqua dest.
  • - Isopropyle alcohol
  • - Glycerine
  • - Beta-Mercaptoethanol
  • - Bromphenolblue


Buffers & gels

  • - Separation buffer (0.5 M Tris, 0.4% SDS, pH = 8.8)
  • - Stacking buffer (0.5 M Tris, 0.4% SDS, pH = 6.6)
  • - Running buffer (0.25 M Tris, 2 M glycine, 1% SDS , pH = 8.3)
  • - Separation gel 12.5% (5 ml Separation buffer, 6.25 ml Rotiphorese®, 3.75 ml Aqua dest., 30 µl TEMED, 30 µl APS (40%))
  • - Stacking gel 4% (3 ml Separation buffer, 1.33 ml Rotiphorese®, 5.67 ml Aqua dest.,20 µl TEMED, 20 µl APS (40%)
  • - 3x Sample buffer (65 mM Tris, 4% SDS, 20% glycerine, 10% beta-Mercaptoethanol, 1 tip of bromphenolblue, pH = 6.75)


Procedure

  1. prepare the separating gel and fill it into the chamber
  2. pour 1 mL isopropyle alcohol on the top of the gel to destroy air bubbles and prevent dehydration
  3. discard the isoprpyl alcohol and pour the prepared stacking gel
  4. stick in the comb
  5. if not used, store the gel in wet cloth (to prevent dehydration) at 4°C

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