Team:Warsaw/Calendar-Main/30 September 2008

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<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>
<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>
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<li>Gel elctrophoresis and <a href="https://2008.igem.org/Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">gel-out</a> of proper bands: 4500 bp - <a href=http://partsregistry.org/wiki/index.php?title=Part:pSB2K3>pSB2K3</a> and 3000 bp - <a href=http://partsregistry.org/Part:BBa_I739204>BBa_I739204 (pACYC177 converted into BioBrick vector)</a>. <a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/30_September_2008#fig1">Fig. 1</a>. </li>
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<li>Gel electrophoresis and <a href="https://2008.igem.org/Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">gel-out</a> of proper bands: 4500 bp - <a href=http://partsregistry.org/wiki/index.php?title=Part:pSB2K3>pSB2K3</a> and 3000 bp - <a href=http://partsregistry.org/Part:BBa_I739204>BBa_I739204 (pACYC177 converted into BioBrick vector)</a>. <a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/30_September_2008#fig1">Fig. 1</a>. </li>
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</ol>
<a name="fig1"><img src="https://static.igem.org/mediawiki/2008/7/79/Go_30_09_2008.jpg
<a name="fig1"><img src="https://static.igem.org/mediawiki/2008/7/79/Go_30_09_2008.jpg
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" width=300/></a> </a><var><b>Fig. 1.</b> EcoRI/BcuI digests of isolated plasmids<br>  
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" width=200/></a> </a><var><b>Fig. 1.</b> EcoRI/BcuI digests of isolated plasmids<br>  
1. Marker<br>
1. Marker<br>
2-3. Digested plasmids BBa_I739204  <br></var>
2-3. Digested plasmids BBa_I739204  <br></var>
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<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). <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#removing">Dephosphorylation</a> (CIAP) of plasmid.</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). <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#removing">Dephosphorylation</a> (CIAP) of plasmid.</li>
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<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 - 3800 bp. <a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/30_September_2008#fig2">Fig. 2</a>.  </li>  
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<li>Gel electrophoresis and <a href="https://2008.igem.org/Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">gel-out</a> of proper band - 3800 bp. <a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/30_September_2008#fig2">Fig. 2</a>.  </li>  
</ol>
</ol>
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<a name="fig2"><img src="https://static.igem.org/mediawiki/2008/8/8f/Go2_30_09_2008na30_09.jpg" width=220/></a> </a><var><b>Fig. 2.</b> BamHI/PstI digests of isolated plasmid<br>  
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<a name="fig2"><img src="https://static.igem.org/mediawiki/2008/8/8f/Go2_30_09_2008na30_09.jpg" width=150/></a> </a><var><b>Fig. 2.</b> BamHI/PstI digests of isolated plasmid<br>  
1. Marker<br>
1. Marker<br>
2. Digested plasmid pACYC_OmpA_omega_deltaA_alpha<br></var>
2. Digested plasmid pACYC_OmpA_omega_deltaA_alpha<br></var>
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<h4>Michał K.</h4>
<h4>Michał K.</h4>
<ol>
<ol>
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<li><a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">Digest</a> of omega_linker 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 omega_linker fragment (PCR from previous day) with NdeI and SacI (BamHI buffer).</li>

Latest revision as of 18:20, 28 October 2008

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Mutagenesis of protein A

Paweł

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

Preparation of vectors for Biobricks

Michał K., Piotr

  1. Isolation of pSB2K3 and BBa_I739204 (pACYC177 converted into BioBrick vector) plasmids.
  2. Digest of isolated plasmids with EcoRI and BcuI (BamHI buffer). Dephosphorylation (CIAP) of plasmids.
  3. Inoculation of bacteria received from iGEM HQs, carrying pSB2K3 and BBa_I739204 (pACYC177 converted into BioBrick vector) plasmids.
  4. Gel electrophoresis and gel-out of proper bands: 4500 bp - pSB2K3 and 3000 bp - BBa_I739204 (pACYC177 converted into BioBrick vector). Fig. 1.
Fig. 1. EcoRI/BcuI digests of isolated plasmids
1. Marker
2-3. Digested plasmids BBa_I739204
4. Digested plasmid psB2K3

Preparation of OmpA-linker-omega-linker (BBa_K103016)

Michał K.

  1. Digest of OmpA-linker-omega-linker (BBa_K103016) PCR product with BglII and PstI (Orange buffer).
  2. Clean-up of above digest reaction.
  3. Digest of pACYC177+OmpA_omega_deltaA_alpha with BamHI and PstI (BamHI buffer). Dephosphorylation (CIAP) of plasmid.
  4. Gel electrophoresis and gel-out of proper band - 3800 bp. Fig. 2.
Fig. 2. BamHI/PstI digests of isolated plasmid
1. Marker
2. Digested plasmid pACYC_OmpA_omega_deltaA_alpha

Preparation of OmpA-linker (BBa_K103006)

Piotr

  1. E. coli TOP10 transformation with overnight ligation pSB1A3+OmpA-linker (BBa_K103006).
  2. Plating on LB + ampicillin.

Preparation of omega_linker under PT7 (BBa_K103020)

Michał K.

  1. Digest of omega_linker fragment (PCR from previous day) with NdeI and SacI (BamHI buffer).
  2. Clean-up of above digest reaction.

Preparation of Z(BBa_K103004)

Piotr

  1. E. coli TOP10 transformation with overnight ligations pSB1A3 + Z(BBa_K103004).
  2. Plating on LB + ampicillin.

Preparation of OmpA_linker_omega_linker under Plac (BBa_K103018)

Michał K.

Overnight digest of purified BBa_K103018 fragment (from previous day) with EcoRI (EcoRI buffer). DNA ends blunting with Klenow fragment.


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