Team:Warsaw/Calendar-Main/3 June 2008

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<h3>Preparation of pMPMT5-AID+AIDT7 construct</h3>
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<h3>Preparation of pMPMT5-AID+AIDT7 construct<br>
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<h4>Michał K.:</h4>
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Michał K.</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</a> of DNA from proper band (3300 bp). </li></ol><br>
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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 (3300 bp). </p>
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<h3>PCR results of translational fusion for the pMPMT5-AID+AIDT7 construct.<br> Lane 1 - 25 cycles, lane 2 - 20 cycles.</h3>
 
<img src="https://static.igem.org/mediawiki/2008/0/08/Tranlacyjna_pcr_29_05_2008.jpg">
<img src="https://static.igem.org/mediawiki/2008/0/08/Tranlacyjna_pcr_29_05_2008.jpg">
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<var>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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<h3>Preparation of pMPMT5+T7 construct<br>
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Piotr:</h3>
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<h3>Preparation of pMPMT5+T7 construct</h3>
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<h4>Piotr:</h4>
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<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest">Digest</a> of pMPMT5-AID+T7 (transcription fusion) with EcoRHI and HindIII (2x Tango buffer).</li>
<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest">Digest</a> of pMPMT5-AID+T7 (transcription fusion) with EcoRHI and HindIII (2x Tango buffer).</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><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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<img src="https://static.igem.org/mediawiki/2008/a/a4/UW_AAT.jpg" width="500"/>
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<img src="https://static.igem.org/mediawiki/2008/f/f0/UW_T.jpg" width="440"/>
<img src="https://static.igem.org/mediawiki/2008/f/f0/UW_T.jpg" width="440"/>

Revision as of 14:51, 9 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 (3300 bp).

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:

  1. Digest of pMPMT5-AID+T7 (transcription fusion) with EcoRHI 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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