Team:Warsaw/Calendar-Main/28 May 2008

From 2008.igem.org

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<html><h3>Preparation of pMPMT5-AID+T7 and pMPMT5+AID-T7</h3>
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<h4>Michał K.:</h4>
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<h3>Rifampicin test #3</h3>
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<h4>Michał L., Ewa, Marcin</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 transcription fusion.</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 <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pMPM-T5-AID>pMPM-T5+AID</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 translation fusion.</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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<li> Control <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest">digest</a> of pMPM-T5 with transcription fusion with EcoRI (EcoRI buffer).</li>
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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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<li> Control <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest">digest</a> of pMPM-T5 with translation fusion with BamHI (BamHI buffer).</li>
 
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<li> Choice of proper clones and preparation for sequencing.</li>
 
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</ol></p>
 
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<h4>Piotr:</h4>
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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>
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<h4>Michał K.</h4>
<p><ol>
<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> <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> 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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<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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<a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pTrc99A-AID>pTrc99A+AID</a></li></ol></p>
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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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</html>
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<h3>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>
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Digest was conducted with BamHI</h3>
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<center>https://static.igem.org/mediawiki/2008/6/62/Trawienie_06_05_2008.jpg</center>
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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>
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<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>
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<li> Choice of proper clones and preparation for sequencing.</li>
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</ol></p>
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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>
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<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>
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Digest was conducted with BamHI</var>
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<div align=center>
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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 transcriptional fusion; <br>7 - pMPMT5-AID; all plasmids digested with EcoRI. </var>
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</html>

Latest revision as of 16:41, 28 October 2008

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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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