Team:Warsaw/Calendar-Main/17 July 2008

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<h3>Cloning of protein Z DNA to OmpA constructs</h3><h4>Michał K.</h4>
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<li> <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">Digest</a> and <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#removing">dephosphorylation (with CIAP)</a> of <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-alpha>pACYC177+OmpA_alpha</a> with SacI and NotI (BamHI buffer). </li>
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<li> Gel eloctrophoresis and <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">gel-out</a> (4300 bp band). </li>
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<a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#ligation">Ligation</a> of isolated DNA fragment and Z DNA fragment isolated on <a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/14_July_2008">14 July</a>. </li>
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<li> <a href="https://2008.igem.org/Wiki/Team:Warsaw/protocols#electrotransform">Transformation</a> of <i>E. coli</i> <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>
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<p>'''Cloning of protein A DNA to OmpA constructs'''<br>
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<h3>Cloning of protein Z DNA to <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pET15b%2BOmpA-omega>pET15b-OmpA-omega</a> in place of OmpA</h3><h4>Paweł</h4>
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1. Digestion 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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<p><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/pET15b%2BOmpA-omega>pET15b-OmpA-omega</a> and Z (in <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pGeneart_Z>Geneart vector</a>) with NdeI and NotI (Orange buffer).</li>  
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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/index.php?title=Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">Gel-out</a> of Z (~200 bp band).</li>
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3. Ligation of A DNA fragment with both pACYC177 vectors. <br>
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<li>Overnight <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#ligation">ligation</a> of Z into digested <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pET15b%2BOmpA-omega>pET15b-OmpA-omega</a>.</li>
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4. Transformation of E. coli TOP10 strain with ligations. <br>
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5. Transformants plating on LB + kanamycin.
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Latest revision as of 09:10, 14 October 2008

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Cloning of protein Z DNA to OmpA constructs

Michał K.

  1. Digest and dephosphorylation (with CIAP) of pACYC177+OmpA_alpha with SacI and NotI (BamHI buffer).
  2. Gel eloctrophoresis and gel-out (4300 bp band).
  3. Ligation of isolated DNA fragment and Z DNA fragment isolated on 14 July.
  4. Transformation of E. coli TOP10 strain with ligation.
  5. Transformants plating on LB + kanamycin.



Cloning of protein Z DNA to pET15b-OmpA-omega in place of OmpA

Paweł

  1. Digest of pET15b-OmpA-omega and Z (in Geneart vector) with NdeI and NotI (Orange buffer).
  2. Gel-out of Z (~200 bp band).
  3. Overnight ligation of Z into digested pET15b-OmpA-omega.

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