Team:Warsaw/Calendar-Main/3 June 2008

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<h3>Preparation of <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pMPM-T5-AID%2BAID-T7>pMPMT5-AID+AIDT7</a> construct</h3>
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<h4>Michał K.</h4>
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<p>
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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</a> of DNA from proper band (20 cycles - 3300 bp). <a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/3_June_2008#fig1">Fig. 1</a>. </p>
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<a name="fig1"><img src="https://static.igem.org/mediawiki/2008/0/08/Tranlacyjna_pcr_29_05_2008.jpg" width=170></a>
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<var><b>Fig. 1.</b> PCR results of translational fusion for the pMPMT5-AID+AIDT7 construct.<br> Lane 1 - 25 cycles, lane 2 - 20 cycles.</var>
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<p>1. DNA (PCR products) gel electrophoresis and isolation of DNA from proper bands<br>
 
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2. Digestion of DNA with HindIII and SalI<br>
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<h3>Preparation of <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pMPM-T5-T7>pMPMT5+T7</a> construct</h3>
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<h4>Piotr, Weronika</h4>
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3. Digestion of pMPM-T5 + AID with HindIII and SalI<br>
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<p><ol>
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4. Clean-up of products of p.2 and p.3 reactions<br>
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<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest">Digest</a> of <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pMPM-T5%2BAID%2BT7>pMPMT5-AID+T7 </a>(transcriptional fusion) with EcoRI and HindIII (2x Tango buffer).</li>
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<li>Use of T4 polymerase for <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#blunting"> blunting</a> (3 hr of incubation).</li>
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<li>DNA gel electrophoresis.</li>
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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</a> from proper band (7000 bp).</li>
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<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#ligation">Ligation</a> of isolated DNA (<a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pMPM-T5-T7>pMPM-T5 + T7 RNA-polymerase</a>).</li>
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<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#chemotransform">Transformation</a> of <i>E.coli</i> <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top10">TOP10</a> with ligation product.</li>
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<li>Plating transformants on LB + tetracycline.</li>
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</ol></p><br>
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5. Ligation of p.4 products (pMPM-T5 + AID transcription fusion with AID-T7 RNA-polymerase translation fusion)<br>
 
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6. Transformation of E.coli TOP10 with ligation product<br>
 
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7. Plating transformants on LB+tetracycline<br>
 
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8. Digestion of pMPM-T5 + transcription fusion with EcoRHI and HindIII<br>
 
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9. Use of T4 polymerase for blunting<br>
 
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10. DNA gel electrophoresis<br>
 
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11. Isolation of DNA from proper band<br>
 
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12. Ligation of product p.11 (pMPM-T5 + T7 RNA-polymerase)<br>
 
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13. Transformation of E.coli TOP10 with ligation product<br>
 
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14. Plating transformants on LB + tetracycline</p>
 
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Latest revision as of 16:47, 28 October 2008

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Preparation of pMPMT5-AID+AIDT7 construct

Michał K.

DNA (PCR products from previous day) gel electrophoresis and isolation of DNA from proper band (20 cycles - 3300 bp). Fig. 1.

Fig. 1. PCR results of translational fusion for the pMPMT5-AID+AIDT7 construct.
Lane 1 - 25 cycles, lane 2 - 20 cycles.

Preparation of pMPMT5+T7 construct

Piotr, Weronika

  1. Digest of pMPMT5-AID+T7 (transcriptional fusion) with EcoRI and HindIII (2x Tango buffer).
  2. Use of T4 polymerase for blunting (3 hr of incubation).
  3. DNA gel electrophoresis.
  4. Isolation of DNA from proper band (7000 bp).
  5. Ligation of isolated DNA (pMPM-T5 + T7 RNA-polymerase).
  6. Transformation of E.coli TOP10 with ligation product.
  7. Plating transformants on LB + tetracycline.

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