Team:Warsaw/Calendar-Main/28 July 2008

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<h3>Checking if omp_omega_A_alpha gives ampicillin resistance<br>
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<h3>Cloning omega-A fusion on <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pKSII%2B>pKS</a> (second attempt)</h3>
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Piotr</h3>
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<h4>Michał L., Ewa, Marcin</h4>
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<p>Sequencing confirms that we have obtained construct with omega-A fusion, but A in our constructs turned out to be truncated and contain only one B-domain (which we called 'delta A'). We inoculated bacteria containing this <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pKSII%2Bomega-deltaA>plasmid</a> to obtain it for futher cloning. </p>
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<ol><li>Inoculation of omp_omega_A_alpha into various IPTG concentrations: 0, 0.1, 0.25, 0.5, 0.75, 1 mmol/mL</li></ol>
 
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<h3>Purification of proteins: Z-alpha and Z-omega</h3>
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<h3>Checking the expression of omp_omega_A_alpha and omp_A_alpha<br>
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<h4>Piotr</h4>
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Piotr</h3>
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<ol><li>Inoculation of omp_omega_A_alpha and omp_A_alpha with inductor (0,5 mmol/mL IPTG) and negative control without IPTG.</li></ol>
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Single transformations of <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#rosetta">Rosetta</a> with plasmids <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pET15b%2BZ-alpha>pET15b+Z-alpha</a> and <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pET15b%2Bhis%2BZ%2Bomega>pET15b+Z-omega</a>. Plating them on LB with chloramphenicol and ampicillin.</p></html>
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<ol><li><a href="https://2008.igem.org/Wiki/Team:Warsaw/protocols#digest">Digest</a> of pACYC177+OmpA_omega_deltaA_alpha (from 25 July) with BamHI and NotI. DNA ends blunting with Klenow fragment (3 hr). </li>
 
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<li>Gel elctrophoresis and <a href="https://2008.igem.org/Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">gel-out</a> of proper band - 4300 bp. </li>
 
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<li><a href="https://2008.igem.org/Wiki/Team:Warsaw/protocols#ligation">Ligation</a> of isolated DNA fragment (1 hr). </li>
 
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<li><a href="https://2008.igem.org/Wiki/Team:Warsaw/protocols#chemotransform">Transformation</a>of E. coli <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top10">TOP10</a> strain with ligation. </li>
 
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<li>Transformants plating on LB + kanamycin. </li></ol>
 
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Cloning omega-A fusion on pKS (second attempt)

Michał L., Ewa, Marcin

Sequencing confirms that we have obtained construct with omega-A fusion, but A in our constructs turned out to be truncated and contain only one B-domain (which we called 'delta A'). We inoculated bacteria containing this plasmid to obtain it for futher cloning.

Purification of proteins: Z-alpha and Z-omega

Piotr

Single transformations of Rosetta with plasmids pET15b+Z-alpha and pET15b+Z-omega. Plating them on LB with chloramphenicol and ampicillin.

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