Team:Warsaw/Calendar-Main/22 August 2008

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<li>Gel elctrophoresis. Chioce of proper clone. </li>
<li>Gel elctrophoresis. Chioce of proper clone. </li>
<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-A>pGeneart+A</a> with NdeI and NotI (Orange buffer), <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pET15b%2BOmpA-alpha>pET15b+OmpA_alpha</a><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#removing"> dephosphorylation with CIAP</a>. </li> </li>
<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-A>pGeneart+A</a> with NdeI and NotI (Orange buffer), <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pET15b%2BOmpA-alpha>pET15b+OmpA_alpha</a><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#removing"> dephosphorylation with CIAP</a>. </li> </li>
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<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 (400 bp for protein A lane and 6200 for pET15b lane). </li>
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<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 (470 bp for protein A lane and 6200 for pET15b lane). </li>
<li> Overnight <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/pET15b%2BA-alpha>pET15b+alpha and A</a>. </li>
<li> Overnight <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/pET15b%2BA-alpha>pET15b+alpha and A</a>. </li>
</ol></p>
</ol></p>

Revision as of 15:34, 24 October 2008

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Tests for ampicillin resistance given by protein added to medium

Piotr

Purification Omp_alpha of antibiotic resistance contamination:
Transformation of E. coli TOP10 strain with pACTC177+OmpA_alpha.

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

Antoni

  1. Isolation of plasmids from cultures inocluated on previous day (pGeneart+A and pET15b+OmpA-alpha).
  2. Control digest of pGeneart+A plasmids with NdeI and NotI (Orange buffer).
  3. Gel elctrophoresis. Chioce of proper clone.
  4. Digest pET15b+OmpA_alpha and pGeneart+A with NdeI and NotI (Orange buffer), pET15b+OmpA_alpha dephosphorylation with CIAP.
  5. Gel electrophoresis of digested plasmids and gel-out of proper bands (470 bp for protein A lane and 6200 for pET15b lane).
  6. Overnight ligation of pET15b+alpha and A.

Cloning of alpha to pACYC177+OmpA_A_omega and pACYC177+OmpA_Z_omega

Michał K.

  1. Ligation of digested vectors from 14 August and alpha DNA fragment from 19 August (more vectors added).
  2. Electroporation of E. coli TOP10 with ligations products.
  3. Transformants plating on LB + kanamycin.

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