Team:Warsaw/Calendar-Main/30 September 2008

From 2008.igem.org

(Difference between revisions)
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<h3>Preparation of BioBricks</h3>
<h3>Preparation of BioBricks</h3>
<h4>Michał K.</h4>
<h4>Michał K.</h4>
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<ol><li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#plasmid_DNA_isolation">Isolation</a> of HQs(??????) plasmids.</li>
+
<ol><li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#plasmid_DNA_isolation">Isolation</a> of <a href=http://partsregistry.org/wiki/index.php?title=Part:pSB2K3>pSB2K3</a> and <a href=http://partsregistry.org/Part:BBa_I739204>BBa_I739204 (pACYC177 converted into BioBrick vector)</a> plasmids.</li>
<li><a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">Digest</a> of isolated plasmids with EcoRI and BcuI (BamHI buffer). </li>
<li><a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">Digest</a> of isolated plasmids with EcoRI and BcuI (BamHI buffer). </li>
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<li> <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">Digest</a> of pACYC177+OmpA_omega_deltaA_alpha with BamHI and PstI (BamHI buffer). </li>
+
<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/pACYC177%2BompA-omega-A-alpha>pACYC177+OmpA_omega_deltaA_alpha</a> with BamHI and PstI (BamHI buffer). </li>
<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#removing">Dephosphorylation</a> (CIAP) of plasmids.</li>
<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#removing">Dephosphorylation</a> (CIAP) of plasmids.</li>
<li>Gel elctrophoresis and <a href="https://2008.igem.org/Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">gel-out</a> of proper band: ??????. </li>  
<li>Gel elctrophoresis and <a href="https://2008.igem.org/Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">gel-out</a> of proper band: ??????. </li>  
<li><a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">Digest</a> of omega_link fragment (PCR from previous day) with EcoRI and BcuI (BamHI buffer)</li>
<li><a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">Digest</a> of omega_link fragment (PCR from previous day) with EcoRI and BcuI (BamHI buffer)</li>
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<li><a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">Digest</a> of OmpA_omega with BglII and PstI (Orange buffer). </li>
+
<li><a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">Digest</a> of OmpA_omega </a> with BglII and PstI (Orange buffer). </li>
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<li>Overnight <a href="https://2008.igem.org/Wiki/Team:Warsaw/protocols#digest">digest</a> of purified pLac_OmpA_omega fragment (from previous day) with EcoRI (EcoRI buffer). DNA ends <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#blunting">blunting</a> with Klenow fragment. </li>
+
<li>Overnight <a href="https://2008.igem.org/Wiki/Team:Warsaw/protocols#digest">digest</a> of purified pLac_OmpA_omega</a> fragment (from previous day) with EcoRI (EcoRI buffer). DNA ends <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#blunting">blunting</a> with Klenow fragment. </li>
</ol>
</ol>
<h3></h3>
<h3></h3>
<h4>Piotr</h4>
<h4>Piotr</h4>
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<ol><li> <i>E. coli</i> <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top10">TOP10</a> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#chemotransform">transformation</a> with overnight ligations (RFP(??????) + Z and RFP(????) + OmpA_link). </li>
+
<ol><li> <i>E. coli</i> <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top10">TOP10</a> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#chemotransform">transformation</a> with overnight ligations <http://partsregistry.org/wiki/index.php?title=Part:pSB2K3>pSB2K3</a> + <a href=http://partsregistry.org/wiki/index.php?title=Part:BBa_K103004>Z(BBa_K103004)</a> and <http://partsregistry.org/wiki/index.php?title=Part:pSB2K3>pSB2K3</a> + OmpA_link). </li>
<li>Plating on LB + ampicillin.</li>
<li>Plating on LB + ampicillin.</li>
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<li>Inoculation of bacterias received from iGEM HQs (         ).</li>
+
<li>Inoculation of bacteria received from iGEM HQs, carrying <a href=http://partsregistry.org/wiki/index.php?title=Part:pSB2K3>pSB2K3</a> and <a href=http://partsregistry.org/Part:BBa_I739204>BBa_I739204 (pACYC177 converted into BioBrick vector)</a> plasmids.</li>
</ol>
</ol>

Revision as of 00:08, 26 October 2008

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Mutagenesis of protein A

Paweł

Results of sequencing: unfortunately all sequences were wild-type.

Preparation of BioBricks

Michał K.

  1. Isolation of pSB2K3 and BBa_I739204 (pACYC177 converted into BioBrick vector) plasmids.
  2. Digest of isolated plasmids with EcoRI and BcuI (BamHI buffer).
  3. Digest of pACYC177+OmpA_omega_deltaA_alpha with BamHI and PstI (BamHI buffer).
  4. Dephosphorylation (CIAP) of plasmids.
  5. Gel elctrophoresis and gel-out of proper band: ??????.
  6. Digest of omega_link fragment (PCR from previous day) with EcoRI and BcuI (BamHI buffer)
  7. Digest of OmpA_omega with BglII and PstI (Orange buffer).
  8. Clean-up of above digest reactions.
  9. Overnight digest of purified pLac_OmpA_omega fragment (from previous day) with EcoRI (EcoRI buffer). DNA ends blunting with Klenow fragment.

Piotr

  1. E. coli TOP10 transformation with overnight ligations pSB2K3 + Z(BBa_K103004) and pSB2K3 + OmpA_link).
  2. Plating on LB + ampicillin.
  3. Inoculation of bacteria received from iGEM HQs, carrying pSB2K3 and BBa_I739204 (pACYC177 converted into BioBrick vector) plasmids.

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