Team:Warsaw/Calendar-Main/3 June 2008

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<h3>Preparation of T7 constructs<br>Piotr</h3>
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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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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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<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</a> of DNA from proper bands. </li>
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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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<li>Digest of DNA with HindIII and SalI (2x Tango buffer).</li>
 
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<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest">Digest</a> of pMPM-T5 + AID with HindIII and SalI.</li>
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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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<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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<h4>Piotr, Weronika</h4>
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<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#ligation">Ligation</a> 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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<li>Plating transformants on LB+tetracycline.</li>
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<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest">Digest</a> of pMPM-T5 + transcription fusion with EcoRHI and HindIII.</li>
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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 blunting.</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>
<li>DNA gel electrophoresis.</li>
<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 from proper band.</a></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 product p.11 (pMPM-T5 + T7 RNA-polymerase).</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#electrotransform">Transformation</a> of <i>E.coli</i> TOP10 with ligation product.</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>
<li>Plating transformants on LB + tetracycline.</li>
<li>Plating transformants on LB + tetracycline.</li>
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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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