Team:Warsaw/Calendar-Main/2 July 2008

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<h3>Change of the reporter from <A href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pZC320>pZC320</a> with B-galactosidase to GFP or RFP</h3>
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<h4>Piotr, Weronika, Emilia</h4>
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<p>'''Preparation of constructs with OmpA protein fusions'''<br>
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1. PCR on OmpA_linker and linker_alpha  with
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Gel electrophoresis and gel-out of proper bands.
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</li>
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<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#OmpaL_N">OmpaL_N</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AlphaP_XB">AlphaP_XB</a>
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primers (20 cycles, annealing length 1.15 min, annealing temperature 57 degrees)<br>
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2. PCR on OmpA_linker and linker_omega with
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<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#OmpaL_N">OmpaL_N</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#OmegaP_EPB">OmegaP_EPB</a>
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primers (20 cycles, annealing length 1.15 min, annealing temperature 57 degrees)<br>
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3. Gel electrophoresis of PCR products and gel-out of OmpA_alpha and OmpA_omega <br>
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4. Overnight digestion of purified OmpA_alpha and OmpA_omega DNA with NdeI and BamHI <br>
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5. Overnight digestion of pET15b plasmid with NdeI and BamHI, dephosphorylation with CIAP
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Ligation of <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pZC320>pZC320</a> with standard parts: <A href=http://partsregistry.org/wiki/index.php?title=Part:BBa_E0840>BBa_E0840</a>(GFP genetrator) and <a href=http://partsregistry.org/wiki/index.php?title=Part:BBa_J04450>BBa_J04450</a> (RFP generator).
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</li>
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<li>Chemotransformation of E.coli TOP10 with ligation products.</li>
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<li>Plating transformants on LB+Amp30+X-gal+IPTG.</li></ol></p>
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<h3>Preparation of constructs on pET15b: OmpA_alpha and OmpA_omega</h3>
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<h4>Paweł</h4>
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<p><ol>
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<li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_purification_after_enzymatic_reaction">Clean-up</a> of overnight digest.</li>
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<li> <A HREF="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#ligation">Ligation</A> of OmpA_alpha (from Michał K.) and <a href=http://www.emdbiosciences.com/docs/docs/PROT/TB045.pdf>pET15b</a>. </li>
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<li> <A HREF="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#ligation">Ligation</A> of OmpA_omega (from Michał K.) and <a href=http://www.emdbiosciences.com/docs/docs/PROT/TB045.pdf>pET15b</a>.</li>
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<li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#electrotransform">Transformation</a> of <i>E. coli</i> <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htmtop10">TOP10</a> strain with ligation products. </li>
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<li> Transformants plating on LB + ampicillin.</li></ol>
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</p>
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</html>

Latest revision as of 14:05, 28 October 2008

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Change of the reporter from pZC320 with B-galactosidase to GFP or RFP

Piotr, Weronika, Emilia

  1. Gel electrophoresis and gel-out of proper bands.
  2. Ligation of pZC320 with standard parts: BBa_E0840(GFP genetrator) and BBa_J04450 (RFP generator).
  3. Chemotransformation of E.coli TOP10 with ligation products.
  4. Plating transformants on LB+Amp30+X-gal+IPTG.

Preparation of constructs on pET15b: OmpA_alpha and OmpA_omega

Paweł

  1. Clean-up of overnight digest.
  2. Ligation of OmpA_alpha (from Michał K.) and pET15b.
  3. Ligation of OmpA_omega (from Michał K.) and pET15b.
  4. Transformation of E. coli TOP10 strain with ligation products.
  5. Transformants plating on LB + ampicillin.

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