Team:Warsaw/Calendar-Main/29 July 2008

From 2008.igem.org

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<h3>Cloning of omega_A DNA fragment to <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-alpha>pACYC177+OmpA_alpha</a></h3><h4>Michał K.</h4>
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<h3>Cloning of omega_&Delta;A DNA fragment to <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-alpha>pACYC177+OmpA_alpha</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</a> of <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pKSII%2Bomega-A>pKS+omega_A</a> from culture inoculated on previous day.</li>
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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=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pKSII%2Bomega-deltaA>pKS+omega_&Delta;A</a> from culture inoculated on previous day.</li>
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<li><a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest>Digest</a> of <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pKSII%2Bomega-A>pKS+omega_A</a>  with SacI and NotI (BamHI buffer).</li>
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<li><a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest>Digest</a> of <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pKSII%2Bomega-deltaA>pKS+omega_&Delta;A</a>  with SacI and NotI (BamHI buffer).</li>
<li><a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest>Digest</a> (SacI and NotI) and <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#removing">dephosphorylation</a> (CIAP) of <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-alpha>pACYC177+OmpA_alpha</a>. </li>
<li><a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest>Digest</a> (SacI and NotI) and <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#removing">dephosphorylation</a> (CIAP) of <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-alpha>pACYC177+OmpA_alpha</a>. </li>
<li>Gel electrophoresis and <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">gel-out</a> of 4300 bp (<a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-alpha>pACYC177+OmpA_alpha</a>) and 550 bp (omega_A) bands. </li>
<li>Gel electrophoresis and <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">gel-out</a> of 4300 bp (<a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-alpha>pACYC177+OmpA_alpha</a>) and 550 bp (omega_A) bands. </li>

Revision as of 13:01, 25 October 2008

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Cloning of omega_ΔA DNA fragment to pACYC177+OmpA_alpha

Michał K.

  1. Isolation of pKS+omega_ΔA from culture inoculated on previous day.
  2. Digest of pKS+omega_ΔA with SacI and NotI (BamHI buffer).
  3. Digest (SacI and NotI) and dephosphorylation (CIAP) of pACYC177+OmpA_alpha.
  4. Gel electrophoresis and gel-out of 4300 bp (pACYC177+OmpA_alpha) and 550 bp (omega_A) bands.
  5. Ligation of DNA fragments from 2. and 3. (1 hr)
  6. Transformation of E. coli TOP10 strain with ligation.
  7. Transformants plating on LB + kanamycin.

Purification of proteins: Z-alpha and Z-omega

Piotr

pET15b+Z_alpha and pET15b+Z_omega in Rosetta strain inoculated in liquid LB with chloramphenicol and ampicillin.

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