Team:Warsaw/Calendar-Main/26 August 2008

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<h3>Cloning of protein A DNA to pET15b+Omp-alpha plasmid</h3><h4>Antoni</h4>
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<h3>Cloning of protein A DNA to <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pET15b%2BOmpA-alpha>pET15b+OmpA-alpha</a> plasmid</h3><h4>Antoni</h4>
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<li> Colony <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> on colonies from plates with transformations pGeneart+A Primers used:
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<li> Colony <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> on colonies from plates with transformations <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pGeneart-A>pGeneart+A</a> Primers used:
<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AP+NotI">AP+NotI</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AL+SacI">AL+SacI.</a></li>
<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AP+NotI">AP+NotI</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AL+SacI">AL+SacI.</a></li>
<li> Lack of confirmed transformant colonies. </li></ol></p>
<li> Lack of confirmed transformant colonies. </li></ol></p>
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<h3>Cloning of protein Z DNA to pET15b+Omp-alpha plasmid</h3><h4>Antoni</h4>
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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> plasmid</h3><h4>Antoni</h4>
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<li> <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">Digest</a> pET15b+OmpA_alpha and pGeneart+Z with NdeI and NotI (Orange buffer), pET15b+OmpA_alpha<a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#removing"> dephosphorylation with CIAP</a>. </li>  
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<li> <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">Digest</a> <A href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pET15b%2BOmpA-alpha>pET15b+OmpA_alpha</a> and <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pGeneart_Z>pGeneart+Z</a> with NdeI and NotI (Orange buffer), pET15b+OmpA_alpha<a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#removing"> dephosphorylation with CIAP</a>. </li>  
<li> Gel electrophoresis of digested plasmids 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 (200 bp for Z lane and 6200 bp for pET15b lane).  </li>
<li> Gel electrophoresis of digested plasmids 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 (200 bp for Z lane and 6200 bp for pET15b lane).  </li>
<li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#ligation">Ligation</a> of pET15b+alpha and Z. </li>
<li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#ligation">Ligation</a> of pET15b+alpha and Z. </li>
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<h3>Cloning of alpha to pACYC177+OmpA_A_omega and pACYC177+OmpA_Z_omega</h3><h4>Michał K.</h4>
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<h3>Cloning of alpha to <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-A-omega>pACYC177+OmpA_A_omega</a> and <a hrehttps://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-Z-omega>pACYC177+OmpA_Z_omega</a></h3><h4>Michał K.</h4>
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<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 (both potential constructs). </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 (both potential constructs). </li>

Revision as of 11:25, 19 October 2008

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Cloning of protein A DNA to pET15b+OmpA-alpha plasmid

Antoni

  1. Colony PCR on colonies from plates with transformations pGeneart+A Primers used: AP+NotI and AL+SacI.
  2. Lack of confirmed transformant colonies.

Cloning of protein Z DNA to pET15b+OmpA-alpha plasmid

Antoni

  1. Digest pET15b+OmpA_alpha and pGeneart+Z with NdeI and NotI (Orange buffer), pET15b+OmpA_alpha dephosphorylation with CIAP.
  2. Gel electrophoresis of digested plasmids and gel-out of proper bands (200 bp for Z lane and 6200 bp for pET15b lane).
  3. Ligation of pET15b+alpha and Z.
  4. Transformation of E. coli TOP10.
  5. Transformants plating on LB + ampicillin.

Cloning of alpha to pACYC177+OmpA_A_omega and pACYC177+OmpA_Z_omega

Michał K.

  1. Isolation of plasmids from cultures inocluated on previous day (both potential constructs).
  2. Control digest of isolated plasmids with FastBamHI and FastAcc65I (Fast Digest buffer).
  3. Gel electrophoresis - still no proper clones founded.

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