Team:Warsaw/Calendar-Main/17 July 2008

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<p>'''Cloning of protein A DNA to OmpA constructs'''<br>
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<h3>Cloning of protein A DNA to OmpA constructs</h3>
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1. Digest of pKS-A plasmid, pACYC177+OmpA_omega and pACYC177+OmpA_alpha with NotI and SacI (pACYC177 were also dephosporylated with CIAP)<br>
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2. Gel electrophoresis and gel-out of proper bands (420 bp - pKS-A lane, 4050 bp - pACYC177+OmpA_omega lane and 4300 bp pACYC177+OmpA_alpha lane). <br>
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<li> <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">Digest</a> of pKS-A plasmid, pACYC177+OmpA_omega and pACYC177+OmpA_alpha with NotI and SacI (pACYC177 were also dephosporylated with CIAP)</li>
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3. Ligation of A DNA fragment with both pACYC177 vectors. <br>
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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">gel-out</a> of proper bands (420 bp - pKS-A lane, 4050 bp - pACYC177+OmpA_omega lane and 4300 bp pACYC177+OmpA_alpha lane). </li>
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4. Transformation of E. coli TOP10 strain with ligations. <br>
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<li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#ligation">Ligation</a> of A DNA fragment with both pACYC177 vectors.</li>
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5. Transformants plating on LB + kanamycin.  
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<li> Transformation of <i>E. coli</i> <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top">TOP10</a>  strain with ligations. </li>
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<li>Transformants plating on LB + kanamycin. </li></ol>
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<p>Pawel: pET15b vector transformed into TOP10 strain for plasmid purification </p>
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<p>Paweł: pET15b vector transformed into TOP10 strain for plasmid purification </p>
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Revision as of 18:17, 1 October 2008

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Cloning of protein A DNA to OmpA constructs

  1. Digest of pKS-A plasmid, pACYC177+OmpA_omega and pACYC177+OmpA_alpha with NotI and SacI (pACYC177 were also dephosporylated with CIAP)
  2. Gel electrophoresis and gel-out of proper bands (420 bp - pKS-A lane, 4050 bp - pACYC177+OmpA_omega lane and 4300 bp pACYC177+OmpA_alpha lane).
  3. Ligation of A DNA fragment with both pACYC177 vectors.
  4. Transformation of E. coli TOP10 strain with ligations.
  5. Transformants plating on LB + kanamycin.

Paweł: pET15b vector transformed into TOP10 strain for plasmid purification


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