Team:Warsaw/Calendar-Main/22 August 2008

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<h3>Tests for ampicillin resistance given by protein added to medium</h3><h4>Piotr</h4>
<h3>Tests for ampicillin resistance given by protein added to medium</h3><h4>Piotr</h4>
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<p>Purification of OmpA_alpha from antibiotic resistance contamination:<br>
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<p>Purification of <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-alpha>pACYC177+OmpA_alpha</a> from antibiotic resistance contamination:<br>
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  <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#chemotrotransform">Transformation</a> of <i>E. coli</i> <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top10">TOP10</a>  strain with pACTC177+OmpA_alpha. </p>
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  <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#chemotrotransform">Transformation</a> of <i>E. coli</i> <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top10">TOP10</a>  strain with <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-alpha>pACYC177+OmpA_alpha</a>. </p>
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<h3>Cloning of protein A DNA to <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pET15b%2BOmpA-alpha>pET15b+OmpA-alpha</a> plasmid</h3><h4>Antoni</h4>
 
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<p><ol>
 
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<li> Control <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">digest</a> of <A href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pGeneart-A>pGeneart+A</a> plasmids with NdeI and NotI (Orange buffer). </li>
 
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<li>Gel elctrophoresis. Choice of proper clone. </li>
 
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<li> <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">Digest</a> of <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pGeneart-A>pGeneart+A</a> with NdeI and NotI (Orange buffer). </li>
 
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<li> Gel electrophoresis of digested plasmid and <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">gel-out</a> of 470 bp band. </li>
 
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<li> Overnight <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#ligation">ligation</a> of <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pET15b%2BA-alpha>pET15b+alpha and A</a> (pET15b+alpha from <a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/19_August_2008">19 August</a>). </li>
 
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</ol></p>
 
<h3>Cloning of alpha to <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-A-omega>pACYC177+OmpA_A_omega</a> and <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-Z-omega>pACYC177+OmpA_Z_omega</a></h3><h4>Michał K.</h4>
<h3>Cloning of alpha to <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-A-omega>pACYC177+OmpA_A_omega</a> and <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-Z-omega>pACYC177+OmpA_Z_omega</a></h3><h4>Michał K.</h4>
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<li>  Transformants plating on LB + kanamycin.</li></ol>
<li>  Transformants plating on LB + kanamycin.</li></ol>
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<h3>Cloning of protein A DNA to GeneArt plasmid</h3><h4>Antoni</h4><ol>
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<li> Control <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">digest</a> of <A href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pGeneart-A>pGeneart+A</a> plasmids with NdeI and NotI (Orange buffer). </li>
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<li>Gel electrophoresis (<a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/22_August_2008#fig1">Fig. 1.</a>). Choice of proper clone. </li></ol>
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<br>
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<a name=fig1><img src="https://static.igem.org/mediawiki/2008/d/d0/PGA%2BAtraw.jpg" width=150></a>
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<var><b>Fig. 1.</b>Control digest of freshly ligated pKS+A (lanes 1-3). Lane 4 - 0.5&mu;g of DNA ladder mix.  </var>
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<h3>Cloning of protein A DNA to <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pET15b%2BOmpA-alpha>pET15b+OmpA-alpha</a> plasmid</h3><h4>Antoni</h4>
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<p><ol>
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<li> <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">Digest</a> of <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pGeneart-A>pGeneart+A</a> with NdeI and NotI (Orange buffer). </li>
 +
<li> Gel electrophoresis of digested plasmid and <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">gel-out</a> of 470 bp band. </li>
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<li> Overnight <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#ligation">ligation</a> of <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pET15b%2BA-alpha>pET15b+alpha and A</a> (pET15b+alpha from <a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/19_August_2008">19 August</a>). </li>
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</ol></p>

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Tests for ampicillin resistance given by protein added to medium

Piotr

Purification of pACYC177+OmpA_alpha from antibiotic resistance contamination:
Transformation of E. coli TOP10 strain with pACYC177+OmpA_alpha.

Cloning of alpha to pACYC177+OmpA_A_omega and pACYC177+OmpA_Z_omega

Michał K.

  1. Ligation of digested vectors from 14 August and alpha DNA fragment from 19 August (more vectors added).
  2. Electroporation of E. coli TOP10 with ligations products.
  3. Transformants plating on LB + kanamycin.

Cloning of protein A DNA to GeneArt plasmid

Antoni

  1. Control digest of pGeneart+A plasmids with NdeI and NotI (Orange buffer).
  2. Gel electrophoresis (Fig. 1.). Choice of proper clone.

Fig. 1.Control digest of freshly ligated pKS+A (lanes 1-3). Lane 4 - 0.5μg of DNA ladder mix.

Cloning of protein A DNA to pET15b+OmpA-alpha plasmid

Antoni

  1. Digest of pGeneart+A with NdeI and NotI (Orange buffer).
  2. Gel electrophoresis of digested plasmid and gel-out of 470 bp band.
  3. Overnight ligation of pET15b+alpha and A (pET15b+alpha from 19 August).


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