Team:Warsaw/Calendar-Main/3 June 2008

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<h3>Preparation of T7 constructs</h3>
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<h3><small>By Michał K.</small></h3>
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<li>DNA (PCR products from previous day) gel electrophoresis and <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">isolation of DNA from proper bands</a>. </li>
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<li>Digestion of DNA with HindIII and SalI.</li>
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<p>DNA (PCR products from previous day) gel electrophoresis and isolation of DNA from proper bands.<br>
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<li>Digestion of pMPM-T5 + AID with HindIII and SalI.</li>
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<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_purification_after_enzymatic_reaction">Clean-up of reaction products.</a></li>
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Digestion of DNA with HindIII and SalI.<br>
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<li>Ligation of p.4 products (pMPM-T5 + AID transcription fusion with AID-T7 RNA-polymerase translation fusion).</li>
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<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#electrotransform">Transformation</a> of <i>E.coli</i> TOP10 with ligation product.</li>
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Digestion of pMPM-T5 + AID with HindIII and SalI.<br>
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<li>Plating transformants on LB+tetracycline.</li>
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<li>Digestion of pMPM-T5 + transcription fusion with EcoRHI and HindIII.</li>
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Clean-up of products of p.2 and p.3 reactions.<br>
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<li>Use of T4 polymerase for blunting.</li>
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<li>DNA gel electrophoresis.</li>
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Ligation of p.4 products (pMPM-T5 + AID transcription fusion with AID-T7 RNA-polymerase translation fusion).<br>
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<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">Isolation of DNA from proper band.</a></li>
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<li>Ligation of product p.11 (pMPM-T5 + T7 RNA-polymerase).</li>
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Transformation of E.coli TOP10 with ligation product.<br>
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<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#electrotransform">Transformation</a> of <i>E.coli</i> TOP10 with ligation product.</li>
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<li>Plating transformants on LB + tetracycline.</li>
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Plating transformants on LB+tetracycline.<br>
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</ul>
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Digestion of pMPM-T5 + transcription fusion with EcoRHI and HindIII.<br>
+
-
 
+
-
Use of T4 polymerase for blunting.<br>
+
-
 
+
-
DNA gel electrophoresis.<br>
+
-
 
+
-
Isolation of DNA from proper band.<br>
+
-
 
+
-
Ligation of product p.11 (pMPM-T5 + T7 RNA-polymerase).<br>
+
-
 
+
-
Transformation of E.coli TOP10 with ligation product.<br>
+
-
 
+
-
Plating transformants on LB + tetracycline.</p>
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Revision as of 15:13, 26 September 2008

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Preparation of T7 constructs

By Michał K.

  • DNA (PCR products from previous day) gel electrophoresis and isolation of DNA from proper bands.
  • Digestion of DNA with HindIII and SalI.
  • Digestion of pMPM-T5 + AID with HindIII and SalI.
  • Clean-up of reaction products.
  • Ligation of p.4 products (pMPM-T5 + AID transcription fusion with AID-T7 RNA-polymerase translation fusion).
  • Transformation of E.coli TOP10 with ligation product.
  • Plating transformants on LB+tetracycline.
  • Digestion of pMPM-T5 + transcription fusion with EcoRHI and HindIII.
  • Use of T4 polymerase for blunting.
  • DNA gel electrophoresis.
  • Isolation of DNA from proper band.
  • Ligation of product p.11 (pMPM-T5 + T7 RNA-polymerase).
  • Transformation of E.coli TOP10 with ligation product.
  • Plating transformants on LB + tetracycline.

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