Team:Warsaw/Calendar-Main/17 July 2008

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<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">Gel-out</a> of Z (~200 bp band).</li>
<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">Gel-out</a> of Z (~200 bp band).</li>
<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#ligation">Ligation</a> of Z into digested pET15b-OmpA-omega.</li>
<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#ligation">Ligation</a> of Z into digested pET15b-OmpA-omega.</li>
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</ol></p>
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<br>
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<h3>Cloning of protein A DNA to geneart plasmid in place of protein Z DNA<br>Antoni</h3>
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<p><ol>
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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#OmpaL_N">AP_Not</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#OmpaP_link">AL_Sac</a>
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<li> Confirmed transformant colonies inoculated to liquid LB with ampicillin
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</li>
</ol></p>
</ol></p>

Revision as of 21:44, 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

  1. Digest of pET15b-OmpA-omega and Z(in GeneArt vector) with NdeI and NotI.
  2. Gel-out of Z (~200 bp band).
  3. Ligation of Z into digested pET15b-OmpA-omega.


Cloning of protein A DNA to geneart plasmid in place of protein Z DNA
Antoni

  1. Colony PCR on colonies from plates with transformations pGeneart+A Primers used: AP_Not and AL_Sac
  2. Confirmed transformant colonies inoculated to liquid LB with ampicillin


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