Team:Warsaw/Calendar-Main/2 June 2008

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<h3>Piotr:</h3>  
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<h3> Rifampicin test #3</h3>
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<h4>Piotr</h4>
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<ol>
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<table id="result">
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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#top">TOP10</a> with <a href="https://2008.igem.org/Wiki/Team:Warsaw/vectors/pZC320">pZC320</a>.</li>
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<tr><th>Strain</th><th>Inducer</th><th>Number of colonies on rifampicin</th><th>OD</th></tr>
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<tr><th><a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top10">TOP10</a></th><td>none</td><td>9</td><td>2.66</td></tr>
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<tr><th><a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top10">TOP10</a> <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pMPM-T5-AID>pMPM-T5-AID</a></th><td>none</td><td>15</td><td>2.5</td></tr>
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<tr><th><a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top10">TOP10</a> <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pMPM-T5-AID>pMPM-T5-AID</a></th><td>0.1% Arabinose</td><td>42</td><td>2.27</td></tr>
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<tr><th><a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top10">TOP10</a> <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pTrc99A-AID>pTrc99a-AID</a></th><td>none</td><td>12</td><td>2.41</td></tr>
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<tr><th><a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top10">TOP10</a> <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pTrc99A-AID>pTrc99a-AID</a></th><td>0.5 mM IPTG</td><td>35</td><td>1.98</td></tr>
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</table>
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<br>
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<p>Conclusion: AID seems to work, induction of AID may reduces the growth rate</p>
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<li> Plating transformants on LB + ampicillin(30μg/mL) + X-gal + IPTG.</li></ol></p>
 
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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><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> for AID transcriptional fusion with translational fusion AID+T7 RNA-polymerase. <br>
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<h3><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> for AID transcription fusion with translation fusion AID+T7 RNA-polymerase <br>Michał K.:</h3>
 
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<p>
 
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Primers: <br>
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Primers:  
<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AIDlNrH">AIDlNrH</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#T7pXbSal">T7pXbSal</a><br>
<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AIDlNrH">AIDlNrH</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#T7pXbSal">T7pXbSal</a><br>
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Template DNA: translation fusion AID+T7 RNA-polymerase (pMPM-T5)<br>
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Template DNA: translational fusion AID+T7 RNA-polymerase (<a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pMPM-T5%2BAIDT7>pMPMT5+AID-T7</a>)<br>
Elongation temperature: 55°C<br>
Elongation temperature: 55°C<br>
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Time: 4 minutes<br>
Time: 4 minutes<br>
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Number of cycles: 20
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Number of cycles: 20 and 25
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</p>
</p>
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<h3> Blue/white and rifampicin test #1</h3>
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<h4>Michał L., Ewa, Marcin</h4>
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<p>
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<ol>
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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 <a href="https://2008.igem.org/Wiki/Team:Warsaw/vectors/pZC320">pZC320</a>.</li>
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<li> Plating transformants on LB + ampicillin (30 μg/mL) + X-gal + IPTG.</li></ol><br></p>

Latest revision as of 14:51, 26 October 2008

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Rifampicin test #3

Piotr

StrainInducerNumber of colonies on rifampicinOD
TOP10none92.66
TOP10 pMPM-T5-AIDnone152.5
TOP10 pMPM-T5-AID0.1% Arabinose422.27
TOP10 pTrc99a-AIDnone122.41
TOP10 pTrc99a-AID0.5 mM IPTG351.98

Conclusion: AID seems to work, induction of AID may reduces the growth rate

Preparation of pMPMT5-AID+AIDT7 construct

Michał K.

PCR for AID transcriptional fusion with translational fusion AID+T7 RNA-polymerase.
Primers: AIDlNrH and T7pXbSal
Template DNA: translational fusion AID+T7 RNA-polymerase (pMPMT5+AID-T7)
Elongation temperature: 55°C
Time: 4 minutes
Number of cycles: 20 and 25

Blue/white and rifampicin test #1

Michał L., Ewa, Marcin

  1. Transformation of E. coli TOP10 with pZC320.
  2. Plating transformants on LB + ampicillin (30 μg/mL) + X-gal + IPTG.

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