Team:Warsaw/Calendar-Main/30 September 2008

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<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/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>
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<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) <a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/30_September_2008#fig1">Fig. 1</a><a name="fig1">. </li></ol>
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<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></ol>
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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>
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<ol><a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/30_September_2008#fig1">Fig. 1</a><a name="fig1">
<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/Team:Warsaw/Calendar-Main/30_September_2008#fig2">Fig. 2</a><a name="fig2">. </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/Team:Warsaw/Calendar-Main/30_September_2008#fig2">Fig. 2</a><a name="fig2">. </li>

Revision as of 21:16, 26 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.

  1. Isolation of pSB2K3 and BBa_I739204 (pACYC177 converted into BioBrick vector) plasmids.
  2. Digest of isolated plasmids with EcoRI and BcuI (BamHI buffer).

Preparation of BioBricks

Michał K.

    Fig. 1
  1. Digest of pACYC177+OmpA_omega_deltaA_alpha with BamHI and PstI (BamHI buffer) Fig. 2.
  2. Dephosphorylation (CIAP) of plasmids.
  3. Gel elctrophoresis and gel-out of proper band: ??????.
  4. Digest of omega_link fragment (PCR from previous day) with EcoRI and BcuI (BamHI buffer)
  5. Digest of OmpA_omega with BglII and PstI (Orange buffer).
  6. Clean-up of above digest reactions.
  7. 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 pSB1A3 + Z(BBa_K103004) and pSB1A3 + 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.
Fig. 1. Control EcoRI/BcuI digests of isolated plasmids
1. Marker
2-3. digested plasmids BBa_I739204
4. digested plasmid psB2K3 Fig. 2. Control BamHI/PstI digests of isolated plasmids
1. Marker
2. digested plasmid pACYC_OmpA_omega_deltaA_alpha