Team:Warsaw/Calendar-Main/10 July 2008

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<h3>Preparation of constructs with OmpA protein fusions</h3>
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<h3>Preparation of construct <a href="https://2008.igem.org/Wiki/Team:Warsaw/vectors/pKSII+A">pKS with A protein</a></h3>
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<h4>Michał L., Marcin</h4>
<p><ol>
<p><ol>
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<li> Inactivation of restriction enzymes and CIAP.</li>
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<li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#ligation">Ligation</a> of digested PCR product and <a href="https://2008.igem.org/Wiki/Team:Warsaw/vectors/pKSII%2B">pKS</a> for 2h at room temperature.</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.htm#top10">TOP10</a> strain with 7 µl of ligation mix.</li>
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<li> Transformants plating on LB + ampicillin + X-gal + IPTG.</li></ol></p>
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<h3>Cloning of protein Z DNA to <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pET15b%2BOmpA-alpha>pET15b-OmpA-alpha</a> in place of OmpA</h3><h4>Piotr, Antoni</h4>
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<p><ol><li>Result of ligation of <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pET15b%2BOmpA-omega>pET15b-OmpA-alpha </a> with protein Z DNA: 16 colonies grown.</li>
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<li>Each colony cultured overnight in LB + ampicilin.</li>
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</ol>
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</p>
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<h3>Preparation of constructs: OmpA_alpha and OmpA_omega #2</h3>
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<h4> Michał K.</h4><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">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>
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<li> Control <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">digest</a> of isolated plasmids with BamHI and NotI (BamHI buffer).
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<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>
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</li>
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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 ligation products: pACYC177 and OmpA_alpha.</li>
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<li> Gel electrophoresis (<a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/10_July_2008#fig1">Fig. 1.</a>) - we confirmed <a href="https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-omega">pACYC177 + OmpA_omega</a>. We didn't obtain <a href="https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-alpha">pACYC177 + OmpA_alpha</a> probably because of a mistake in plating. </li>
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<li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#ligation">Ligation</a> of <a href="https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177">pACYC177</a> and OmpA_alpha (1 hr).</li>
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<li> Transformation of <i>E. coli</i> <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top10">TOP10</a> strain with ligation products: <a href="https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-alpha">pACYC177 and OmpA_alpha</a>.</li>
<li> Transformants plating on LB + kanamycin.</li>
<li> Transformants plating on LB + kanamycin.</li>
</ol></p>
</ol></p>
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<h3>Cloning of protein Z DNA to OmpA constructs</h3>
 
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<p><ol>
 
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<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>
 
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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 220 bp (for Geneart_Z lane)and 4050 bp (pACYC177+OmpA_omega lane). </li>
 
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<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>
 
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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 ligation. </li>
 
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<li> Transformants plating on LB + kanamycin.</li>
 
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</ol>
 
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</p>
 
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<h3>Preparation of construct pKS with A protein</h3>
 
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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#plasmid_DNA_isolation">Isolation of plasmid DNA</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">digest</a> of isolated plasmid with SacI and NotI; 2 h</li>
 
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<li> Gel electrophoresis of digested DNA</li>
 
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<li> We are glad to find the proper clone ;-) and inoculate liquid LB with ampicillin to freeze bacteria carrying pKS-A construct</li></ol>
 
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</p>
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<a name="fig1"><img src="https://static.igem.org/mediawiki/2008/9/9c/Trawienie_5_th_july.jpg" width=300 /></a><var><b>Fig. 1. </b>Control BamHI and NotI digests  of plasmids, which were used in successful colony PCRs <b></b><br>
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1. Marker<br>
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2. slot 1. from yesterday's Omp_A_alpha colony PCR<br>
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2. slot 14. from yesterday's Omp_A_alpha colony PCR<br>
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2. slot 4. from yesterday's Omp_A_omega colony PCR<br>
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2. slot 10. from yesterday's Omp_A_omega colony PCR<br>
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Latest revision as of 17:54, 26 October 2008

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Preparation of construct pKS with A protein

Michał L., Marcin

  1. Inactivation of restriction enzymes and CIAP.
  2. Ligation of digested PCR product and pKS for 2h at room temperature.
  3. Transformation of E. coli TOP10 strain with 7 µl of ligation mix.
  4. Transformants plating on LB + ampicillin + X-gal + IPTG.

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

Piotr, Antoni

  1. Result of ligation of pET15b-OmpA-alpha with protein Z DNA: 16 colonies grown.
  2. Each colony cultured overnight in LB + ampicilin.

Preparation of constructs: OmpA_alpha and OmpA_omega #2

Michał K.

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

Fig. 1. Control BamHI and NotI digests of plasmids, which were used in successful colony PCRs
1. Marker
2. slot 1. from yesterday's Omp_A_alpha colony PCR
2. slot 14. from yesterday's Omp_A_alpha colony PCR
2. slot 4. from yesterday's Omp_A_omega colony PCR
2. slot 10. from yesterday's Omp_A_omega colony PCR

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