Team:Warsaw/Calendar-Main/10 July 2008

From 2008.igem.org

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<p><ol>
<p><ol>
<li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#plasmid_DNA_isolation">Isolation of plasmids</a> from cultures inocluated on previous day. </li>
<li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#plasmid_DNA_isolation">Isolation of plasmids</a> from cultures inocluated on previous day. </li>
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<li> Control <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">digestion</a> of isolated plasmids with BamHI and NotI (we confirmed pCACYC177 + OmpA_omega). We didn't obtain pCACYC177 + OmpA_alpha probably because of a mistake in plating. </li>
+
<li> Control <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">digest</a> of isolated plasmids with BamHI and NotI (we confirmed pCACYC177 + OmpA_omega). We didn't obtain pCACYC177 + OmpA_alpha probably because of a mistake in plating. </li>
<li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#ligation">Ligation</a> of pACYC177 and OmpA_alpha (1 hr)</li>
<li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#ligation">Ligation</a> of pACYC177 and OmpA_alpha (1 hr)</li>
<li> Transformation of <i>E. coli</i> <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top">TOP10</a> strain with ligation products: pACYC177 and OmpA_alpha.</li>
<li> Transformation of <i>E. coli</i> <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top">TOP10</a> strain with ligation products: pACYC177 and OmpA_alpha.</li>
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<h3>Cloning of protein Z DNA to OmpA constructs</h3>
<h3>Cloning of protein Z DNA to OmpA constructs</h3>
<p><ol>
<p><ol>
-
<li> <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">Digestion</a> of Geneart_Z and pACYC177+OmpA_omega with SacI and NotI (pACYC177 was also dephosphorylated with CIAP)(3 hr).</li>
+
<li> <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">Digest</a> of Geneart_Z and pACYC177+OmpA_omega with SacI and NotI (pACYC177 was also dephosphorylated with CIAP)(3 hr).</li>
<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 220 bp (for Geneart_Z lane)and 4050 bp (pACYC177+OmpA_omega lane). </li>
<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 220 bp (for Geneart_Z lane)and 4050 bp (pACYC177+OmpA_omega lane). </li>
<li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#ligation">Ligation</a> of pACYC177+OmpA_omega and Z (1 hr). </li>
<li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#ligation">Ligation</a> of pACYC177+OmpA_omega and Z (1 hr). </li>
Line 24: Line 24:
<p><ol>
<p><ol>
<li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#plasmid_DNA_isolation">Isolation of plasmid DNA</a> from cultures inocluated on previous day. </li>
<li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#plasmid_DNA_isolation">Isolation of plasmid DNA</a> from cultures inocluated on previous day. </li>
-
<li> Control <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">digestion</a> of isolated plasmid with SacI and NotI; 2 h</li>
+
<li> Control <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">digest</a> of isolated plasmid with SacI and NotI; 2 h</li>
<li> Gel electrophoresis of digested DNA</li>
<li> Gel electrophoresis of digested DNA</li>
<li> We are glad to find the proper clone ;-) and inoculate liquid LB with ampicillin to freeze bacteria carrying pKS-A construct</li></ol>
<li> We are glad to find the proper clone ;-) and inoculate liquid LB with ampicillin to freeze bacteria carrying pKS-A construct</li></ol>

Revision as of 17:59, 1 October 2008

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Preparation of constructs with OmpA protein fusions

  1. Isolation of plasmids from cultures inocluated on previous day.
  2. Control digest of isolated plasmids with BamHI and NotI (we confirmed pCACYC177 + OmpA_omega). We didn't obtain pCACYC177 + OmpA_alpha probably because of a mistake in plating.
  3. Ligation of pACYC177 and OmpA_alpha (1 hr)
  4. Transformation of E. coli TOP10 strain with ligation products: pACYC177 and OmpA_alpha.
  5. Transformants plating on LB + kanamycin.

Cloning of protein Z DNA to OmpA constructs

  1. Digest of Geneart_Z and pACYC177+OmpA_omega with SacI and NotI (pACYC177 was also dephosphorylated with CIAP)(3 hr).
  2. Gel electrophoresis and gel-out of proper bands 220 bp (for Geneart_Z lane)and 4050 bp (pACYC177+OmpA_omega lane).
  3. Ligation of pACYC177+OmpA_omega and Z (1 hr).
  4. Transformation of E. coli TOP10 strain with ligation.
  5. Transformants plating on LB + kanamycin.

Preparation of construct pKS with A protein

  1. Isolation of plasmid DNA from cultures inocluated on previous day.
  2. Control digest of isolated plasmid with SacI and NotI; 2 h
  3. Gel electrophoresis of digested DNA
  4. We are glad to find the proper clone ;-) and inoculate liquid LB with ampicillin to freeze bacteria carrying pKS-A construct

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