Team:Warsaw/Calendar-Main/27 August 2008

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<h3>Tests for ampicillin resistance given by protein added to medium</h3><h4>Piotr</h4>
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<h3>Cloning of protein Z DNA to pET15b+Omp-alfa plasmid</h3><h4>Antoni</h4>
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Results of growth from previous day
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<li> Colony PCR on colonies from plates with transformations pGeneart+A Primers used:
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<table id="result">
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<tr><th>OmpA variant ("hunter")</th><th>"Prey" variant</th><th>Growth with prey</th><th>Growth without prey</th></tr>
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<tr><th>OmpA-alpha</th><td>His+Z+omega</td><td>+</td><td>-</td></tr>
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<tr><th>OmpA-alpha</th><td>His+Z+alfa</td><td>-</td><td>-</td></tr>
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<tr><th>OmpA-A-alpha</th><td>His+Z+omega</td><td>+</td><td>-</td></tr>
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</table>
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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>Michał K.</h4>
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<ol><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> and <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pET15b%2BOmpA-alpha>pET15b+OmpA-alpha</a> with NdeI and NotI (Orange buffer), pET15b was also <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#removing"> dephosphorylated</a>. </li>  
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<li> Gel electrophoresis of digested plasmids (<a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/27_August_2008#fig1">Fig. 1.</a>) and <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">gel-out</a> of proper bands (400 bp for A lane and 6200 bp for pET15b lane).  </li>
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<li> <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>. </li>
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<li> Transformation of <i>E. coli</i> <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top10">TOP10</a>. </li>
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<li> Transformants plating on LB + ampicillin.</li>
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<a name="fig1"><img src="
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https://static.igem.org/mediawiki/2008/1/1b/Go29.jpg" width=300/></a> <var><b>Fig. 1.</b> NdeI/NotI digest of plasmid pGeneart+A<br>
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1. Marker<br>
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2. Digested plasmid pGeneart+A<br></var>
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<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AP+NotI">AP+NotI</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AL+SacI">AL+SacI</a>
 
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<li> Confirmed transformant colonies inoculated to liquid LB with ampicillin
 
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Latest revision as of 20:56, 26 October 2008

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Tests for ampicillin resistance given by protein added to medium

Piotr

Results of growth from previous day

OmpA variant ("hunter")"Prey" variantGrowth with preyGrowth without prey
OmpA-alphaHis+Z+omega+-
OmpA-alphaHis+Z+alfa--
OmpA-A-alphaHis+Z+omega+-

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

Michał K.

  1. Digest of pGeneart+A and pET15b+OmpA-alpha with NdeI and NotI (Orange buffer), pET15b was also dephosphorylated.
  2. Gel electrophoresis of digested plasmids (Fig. 1.) and gel-out of proper bands (400 bp for A lane and 6200 bp for pET15b lane).
  3. Ligation of pET15b-Alpha and A.
  4. Transformation of E. coli TOP10.
  5. Transformants plating on LB + ampicillin.
    6. Fig. 1. NdeI/NotI digest of plasmid pGeneart+A
    1. Marker
    2. Digested plasmid pGeneart+A




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