Team:Warsaw/Calendar-Main/19 May 2008

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<html><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>
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<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> - <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pMPM-T5%2BAIDT7>translational fusion</a>: AID + T7 RNA-polymerase<br>
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<p>Michał K.:</p>
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Primers: <br>
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<p>1. Gel electrophoresis (<a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/16_May_2008">friday</a> PCR's) and DNA <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">isolation</a> from proper bands.</p>
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<li><a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AIDlNrH">AIDlNrH</a></li>
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<li><a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#T7pXbSal">T7pXbSal</a></li>
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<p>2. PCR - translation fusion: AID + T7 RNA-polimerase</p>
 
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Primers:
 
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Template DNA: purified PCR products from <a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/16_May_2008">16 May</a> - AID and T7 RNA-polymerase for <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pMPM-T5%2BAIDT7>translational fusion</a> <br>
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<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AIDlNrH">AIDlNrH</a> and  
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<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#T7pXbSal">T7pXbSal</a>
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Template DNA: AID for translation fusion and T7 RNA-polimerase for translation fusion
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Annealing temperature : 73&deg;C <br>
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Elongation time: 4 minutes <br>
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Annealing temperature: 55 °C
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20 cycles <br></li>
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<li> 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 proper band (3300 bp). <a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/19_May_2008#fig1">Fig. 1</a>.</li>
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<li>Electrophoresis to estimate the concentration of isolated DNA.</li></ol></p>
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Annealing time: 4 minutes
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<a name="fig1"><img src="https://static.igem.org/mediawiki/2008/e/e5/Pcr_fusion_WAW.jpg" width=190/></a>
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<var><b>Fig. 1.</b> 1-DNA ladder; 2-PCR product-translational fusion</var>
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20 cycles
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<h3>Rifampicin test #1</h3>
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<h4>Michał L., Ewa, Marcin</h4>
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3. Gel electrophoresis and DNA isolation from proper band (translation fusion: AID + T7 RNA-polimerase)
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<p>Overnight culture of the following strains:<br>
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<a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top10">Top10</a><br>
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<a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top10">Top10</a> + 0.5 mM IPTG<br>
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and <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top10">Top10</a> carrying:<br>
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<a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pMPM-T5-AID>pMPM-T5-AID</a> + Glc <br>
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<a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pTrc99A-AID>pTrc99a-AID</a> + Glc <br>
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<a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pMPM-T5-AID>pMPM-T5-AID</a> + 0.01% L-Ara<br>
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<a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pTrc99A-AID>pTrc99a-AID</a> + 0.5 mM IPTG<br>
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Latest revision as of 16:37, 28 October 2008

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

Michał K.

  1. PCR - translational fusion: AID + T7 RNA-polymerase
    Primers:
    Template DNA: purified PCR products from 16 May - AID and T7 RNA-polymerase for translational fusion
    Annealing temperature : 73°C
    Elongation time: 4 minutes
    20 cycles
  2. Gel electrophoresis and isolation of proper band (3300 bp). Fig. 1.
  3. Electrophoresis to estimate the concentration of isolated DNA.

Fig. 1. 1-DNA ladder; 2-PCR product-translational fusion

Rifampicin test #1

Michał L., Ewa, Marcin

Overnight culture of the following strains:
Top10
Top10 + 0.5 mM IPTG
and Top10 carrying:
pMPM-T5-AID + Glc
pTrc99a-AID + Glc
pMPM-T5-AID + 0.01% L-Ara
pTrc99a-AID + 0.5 mM IPTG

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