Team:Warsaw/Calendar-Main/28 May 2008

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<html><h3>Preparation of pMPMT5-AID+T7 and pMPMT5+AID-T7</h3>
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<h3>Rifampicin test #3</h3>
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<h4>Michał L., Ewa, Marcin</h4>
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<li> Inoculation of liquid LB with E.coli <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top10">TOP10</a> carrying <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pMPM-T5-AID>pMPM-T5+AID</a> </li>
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<li> Inoculation of liquid LB with E.coli <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top10">TOP10</a> carrying
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<a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pTrc99A-AID>pTrc99A+AID</a></li></ol></p><br>
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<h3>Preparation of <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pMPM-T5%2BAID%2BT7>pMPMT5-AID+T7</a> and <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pMPM-T5%2BAIDT7>pMPMT5+AID-T7</a></h3>
<h4>Michał K.</h4>
<h4>Michał K.</h4>
<p><ol>
<p><ol>
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<li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#plasmid_DNA_isolation">Isolation</a> of hypothetical pMPM-T5 with transcriptional fusion.</li>
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<li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#plasmid_DNA_isolation">Isolation</a> of hypothetical <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pMPM-T5%2BAID%2BT7>pMPM-T5 with transcriptional fusion</a>.</li>
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<li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#plasmid_DNA_isolation">Isolation</a> of hypothetical pMPM-T5 with translational fusion.</li>
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<li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#plasmid_DNA_isolation">Isolation</a> of hypothetical <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pMPM-T5%2BAIDT7>pMPM-T5 with translational fusion</a>.</li>
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<li> Control <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest">digest</a> of pMPM-T5 with transcriptional fusion with EcoRI (EcoRI buffer).</li>
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<li> Control <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>pMPM-T5 with transcriptional fusion</a> with EcoRI (EcoRI buffer).</li>
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<li> Control <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest">digest</a> of pMPM-T5 with translational fusion with BamHI (BamHI buffer).</li>
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<li> Control <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%2BAIDT7>pMPM-T5 with translational fusion</a> with BamHI (BamHI buffer).</li>
<li> Gel electrophoresis (<a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/28_May_2008#fig1">Fig. 1</a> and <a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/28_May_2008#fig2">Fig. 2</a>). </li>
<li> Gel electrophoresis (<a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/28_May_2008#fig1">Fig. 1</a> and <a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/28_May_2008#fig2">Fig. 2</a>). </li>
<li> Choice of proper clones and preparation for sequencing.</li>
<li> Choice of proper clones and preparation for sequencing.</li>
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<h3>Rifampicin test</h3>
 
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<h4>Michał L., Ewa, Marcin</h4>
 
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<p><ol>
 
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<li> Inoculation of liquid LB with E.coli <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top10">TOP10</a> carrying pMPM-T5+AID </li>
 
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<li> Inoculation of liquid LB with E.coli <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top10">TOP10</a> carrying
 
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<a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pTrc99A-AID>pTrc99A+AID</a></li></ol></p><br>
 
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<center><a name="fig1"><img src="https://static.igem.org/mediawiki/2008/6/62/Trawienie_06_05_2008.jpg"/></a></center>
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<center><a name="fig1"><img src="https://static.igem.org/mediawiki/2008/6/62/Trawienie_06_05_2008.jpg" width=190/></a></center>
<var><b>Fig. 1.</b> Here we found the clone with translational fusion on pMPM. These are different isolations in each lane. We have been working with the AT6 ever since. <br>
<var><b>Fig. 1.</b> Here we found the clone with translational fusion on pMPM. These are different isolations in each lane. We have been working with the AT6 ever since. <br>
Digest was conducted with BamHI</var>
Digest was conducted with BamHI</var>
<div align=center>
<div align=center>
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<a name="fig1"><img src="https://static.igem.org/mediawiki/2008/d/d6/Translation_fusion_digest_Eco.jpg"width=300/></a></div>
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<a name="fig2"><img src="https://static.igem.org/mediawiki/2008/d/d6/Translation_fusion_digest_Eco.jpg"width=250/></a></div>
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<var><b>Fig. 2.</b> 2 to 6 - Control digests of few clones with transcription fusion; <br>7 - pMPMT5-AID; all plasmids digested with EcoRI. </var>
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<var><b>Fig. 2.</b> 2 to 6 - Control digests of few clones with transcriptional fusion; <br>7 - pMPMT5-AID; all plasmids digested with EcoRI. </var>
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Rifampicin test #3

Michał L., Ewa, Marcin

  1. Inoculation of liquid LB with E.coli TOP10 carrying pMPM-T5+AID
  2. Inoculation of liquid LB with E.coli TOP10 carrying pTrc99A+AID


Preparation of pMPMT5-AID+T7 and pMPMT5+AID-T7

Michał K.

  1. Isolation of hypothetical pMPM-T5 with transcriptional fusion.
  2. Isolation of hypothetical pMPM-T5 with translational fusion.
  3. Control digest of pMPM-T5 with transcriptional fusion with EcoRI (EcoRI buffer).
  4. Control digest of pMPM-T5 with translational fusion with BamHI (BamHI buffer).
  5. Gel electrophoresis (Fig. 1 and Fig. 2).
  6. Choice of proper clones and preparation for sequencing.

Fig. 1. Here we found the clone with translational fusion on pMPM. These are different isolations in each lane. We have been working with the AT6 ever since.
Digest was conducted with BamHI
Fig. 2. 2 to 6 - Control digests of few clones with transcriptional fusion;
7 - pMPMT5-AID; all plasmids digested with EcoRI.



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