Team:Warsaw/Calendar-Main/25 August 2008

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<h3>Cloning of protein A DNA to pET15b+OmpA-alfa plasmid in place of OmpA<br>Antoni</h3>
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<h3>Tests for ampicillin resistance given by protein added to medium</h3><h4>Piotr</h4>
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<p> <ol><li>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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Inoculation of <i>E. coli</i> <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top10">TOP10</a>  carrying pACTC177+OmpA_alpha to liquid LB with kanamycin and with kanamycin and ampicillin.</li>
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<li>Inoculation of TOP10 carrying <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-A-alpha>pACYC177+OmpA_A_alpha</a> and <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-alpha>pACYC177+OmpA-alpha</a> to LB with inductor (0.25 mM IPTG) and kanamycin.</p>
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</li></ol>
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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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<li> Colony PCR on colonies from plates with transformations pGeneart+A Primers used:
 
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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></li>
 
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<li> Lack of confirmed transformant colonies </li></ol></p>
 
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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> strain with <a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/22_August_2008">friday's</a> ligation products. </li>
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<li> Transformants plating on LB + ampicillin.</li>
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<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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<p> Eight colonies from each plate (<a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-alpha-A-omega>pACYC177+OmpA_alpha_A_omega</a> and <A href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BOmpA-alpha-Z-omega>pACYC177+OmpA_alpha_Z_omega</a>) were inoculated to liquid LB with kanamycin.</p>
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Tests for ampicillin resistance given by protein added to medium

Piotr

  1. Purification of pACYC177+OmpA_alpha from antibiotic resistance contamination:
    Inoculation of E. coli TOP10 carrying pACTC177+OmpA_alpha to liquid LB with kanamycin and with kanamycin and ampicillin.
  2. Inoculation of TOP10 carrying pACYC177+OmpA_A_alpha and pACYC177+OmpA-alpha to LB with inductor (0.25 mM IPTG) and kanamycin.

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

Antoni

  1. Transformation of E. coli TOP10 strain with friday's ligation products.
  2. Transformants plating on LB + ampicillin.

Cloning of alpha to pACYC177+OmpA_A_omega and pACYC177+OmpA_Z_omega

Michał K.

Eight colonies from each plate (pACYC177+OmpA_alpha_A_omega and pACYC177+OmpA_alpha_Z_omega) were inoculated to liquid LB with kanamycin.

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