Team:Warsaw/Calendar-Main/29 August 2008

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Protein purification: A-alpha, Z-alpha i Z-omega
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<h3>Purification of proteins: A-alpha</h3>
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Piotr
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<h4>Piotr</h4>
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<p>
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Transformation of <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#rosetta">Rosetta</a> with <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pET15b%2BA-alpha>pET15b+A-alpha</a> plasmid. Plating on LB with chloramphenicol and ampicillin.</p>
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Single transformations of Rosetta with plasmids pET15b+A-alfa, pET15b+Z-alfa and pET15b+Z-omega. Plating them on LB with Cm and Ap.
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<h3>Blue-white screening and rifampicin test in <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#gm">GM2163</a></h3>
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<h4>Michał L.</h4>
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<p>GM2163 competent cells carrying <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pZC320>pZC320</a> were transformed with following plasmids: </p>
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<ul><li><a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pMPM-T5-omega>pMPMT5-omega</a>,</li>
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<li><a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pMPM-T5-AID>pMPMT5-AID</a>,</li>
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<li><a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pMPM-T5%2BAIDT7>pMPMT5-AIDT7</a>, </li>
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<li><a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pMPM-T5%2BAID%2BT7>pMPMT5-AID+T7</a>.</li></ul>
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<p>Transformants were selected on CTAXI plates (LB + Chloramphenicol + Tetracycline + IPTG + Ampicillin 30 μg/mL + X-Gal) because GM2163 are chloramphenicol resistant.</p>
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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>Michał K.</h4>
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<p><ol><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/pET15b%2BA-alpha>pET15b+A-alpha</a> plasmids.</li>
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<li> Control <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">digest</a> of isolated plasmids with XbaI and BamHI (Tango buffer). </li>
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<li>Gel electrophoresis (<a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/29_August_2008#fig1">Fig. 1.</a>). Choice of proper clone. </li>
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</ol></p>
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<a name="fig1"><img src="
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https://static.igem.org/mediawiki/2008/a/a8/Kokodak.jpg" width=300/></a> <var><b>Fig. 1.</b> Control XbaI/BamHI digests of  plasmids isolated from transformants with colony PCR product.<br>
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1. Marker<br>
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2-4. Digested plasmids pET15b+A-alpha <br></var>
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Latest revision as of 20:58, 26 October 2008

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Purification of proteins: A-alpha

Piotr

Transformation of Rosetta with pET15b+A-alpha plasmid. Plating on LB with chloramphenicol and ampicillin.

Blue-white screening and rifampicin test in GM2163

Michał L.

GM2163 competent cells carrying pZC320 were transformed with following plasmids:

Transformants were selected on CTAXI plates (LB + Chloramphenicol + Tetracycline + IPTG + Ampicillin 30 μg/mL + X-Gal) because GM2163 are chloramphenicol resistant.

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

Michał K.

  1. Isolation of pET15b+A-alpha plasmids.
  2. Control digest of isolated plasmids with XbaI and BamHI (Tango buffer).
  3. Gel electrophoresis (Fig. 1.). Choice of proper clone.

Fig. 1. Control XbaI/BamHI digests of plasmids isolated from transformants with colony PCR product.
1. Marker
2-4. Digested plasmids pET15b+A-alpha



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