Team:Warsaw/Calendar-Main/7 October 2008

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<html>
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<h3>Preparation of <a href=http://partsregistry.org/Part:pSB2K3>pSB2K3</a> + _alpha</h3>
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<h3>Preparation of <a href=http://partsregistry.org/Part:BBa_K103009>linker_alpha (BBa_K103009)</a></h3>
-
<h4>Michał K.</h4>
+
<h4>Michał K., Piotr</h4>
<ol>
<ol>
-
<li><a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#plasmid_DNA_isolation>Isolation</a> of plasmid from culture inoculated on previous day (<a href=http://partsregistry.org/Part:pSB2K3>pSB2K3</a> + _alpha).</li>
+
<li><a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#plasmid_DNA_isolation>Isolation</a> of plasmid from culture inoculated on previous day (<a href=http://partsregistry.org/Part:pSB2K3>pSB2K3</a> + <a href=http://partsregistry.org/Part:BBa_K103009>linker_alpha (BBa_K103009)</a>).</li>
-
<li>Control <a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest>digest</a> of isolated plasmid with EcoRI and PstI (Orange buffer). Gel electrophoresis - no proper clones found.</li></ol>
+
<li>Control <a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest>digest</a> of isolated plasmid with EcoRI and PstI (Orange buffer). Gel electrophoresis - no proper clones found. <a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/7_October_2008#fig1">Fig. 1</a>.</li>  
 +
<li>Inoculation of few more colonies which grown on plate with transformation: <a href=http://partsregistry.org/Part:pSB2K3>pSB2K3</a> + <a href=http://partsregistry.org/Part:BBa_K103009>linker_alpha (BBa_K103009)</a> to liquid LB + kanamycin. </li>
 +
<p class="hide"><img src="https://static.igem.org/mediawiki/2008/d/d0/Traw_alfa_omega_07_10_2008.jpg">
 +
<var><b>Fig. 1.</b>Control EcoRI/PstI digest of pSB2K3+linker_omega (BBa_K103013)<br>
 +
1. and 12. Marker<br>
 +
2-9. Control digests of pSB2K3+linker_alpha (BBa_K103009)<br>
 +
10-17. Control digests of pSB2K3+linker_omega (BBa_K103013)<br></var></p>
 +
</ol>
-
<h3>Preparation of BioBricks</h3>
+
<h3>Preparation of <a href=http://partsregistry.org/Part:BBa_K103013>linker_omega (BBa_K103013)</a></h3>
-
<h4>Michał K.</h4>
+
<h4>Michał K., Piotr</h4>
<ol>
<ol>
-
<li><a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#plasmid_DNA_isolation>Isolation</a> of plasmid from culture inoculated on previous day (<a href=http://partsregistry.org/Part:pSB2K3>pSB2K3</a> + _alpha, <a href=http://partsregistry.org/Part:pSB2K3>pSB2K3</a> + _omega and pACYC177 + OmpA_omega_).</li>
+
<li><a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#plasmid_DNA_isolation>Isolation</a> of plasmid from culture inoculated on previous day (<a href=http://partsregistry.org/Part:pSB2K3>pSB2K3</a> + <a href=http://partsregistry.org/Part:BBa_K103013>linker_omega (BBa_K103013)</a>).</li>
-
<li>Control <a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest>digest</a> of isolated plasmid with EcoRI and PstI (Orange buffer). Gel electrophoresis - no proper clones found.</li>
+
<li>Control <a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest>digest</a> of isolated plasmid with EcoRI and PstI (Orange buffer). Gel electrophoresis - no proper clones found.
-
<li> Overnight <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#ligation">ligation</a> of DNA fragments: <a href=http://partsregistry.org/wiki/index.php?title=Part:pSB1A3>pSB1A3</a>+ AID.</li>
+
<a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/7_October_2008#fig1">Fig. 1</a>.</li>
 +
<li>Inoculation of few more colonies which grown on plates with transformation: <a href=http://partsregistry.org/Part:pSB2K3>pSB2K3</a> + <a href=http://partsregistry.org/Part:BBa_K103013>linker_omega (BBa_K103013)</a> to liquid LB + kanamycin. </li>
 +
 
 +
 
</ol>
</ol>
-
<h3></h3>
+
<a name="fig1"><img src="https://static.igem.org/mediawiki/2008/d/d0/Traw_alfa_omega_07_10_2008.jpg"></a>
 +
<var><b>Fig. 1.</b>Control EcoRI/PstI digest of pSB2K3+linker_omega (BBa_K103013)<br>
 +
1. and 12. Marker<br>
 +
2-9. Control digests of pSB2K3+linker_alpha (BBa_K103009)<br>
 +
10-17. Control digests of pSB2K3+linker_omega (BBa_K103013)<br></var></var>
 +
 
 +
 
 +
 
 +
 
 +
 
 +
<h3>Preparation of <a href=http://partsregistry.org/Part:BBa_K103016>OmpA-linker-omega-linker (BBa_K103016)</a></h3>
 +
<h4>Michał K., Piotr</h4>
 +
<ol>
 +
<li><a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#plasmid_DNA_isolation>Isolation</a> of plasmid from culture inoculated on previous day (<a href=http://partsregistry.org/Part:BBa_I739204>pACYC177</a> + <a href=http://partsregistry.org/Part:BBa_K103016>OmpA-linker-omega-linker (BBa_K103016)</a>).</li>
 +
<li>Control <a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest>digest</a> of isolated plasmid with EcoRI and PstI (Orange buffer). Gel electrophoresis - no proper clones found.
 +
<a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/7_October_2008#fig2">Fig. 2</a>.</li>
 +
<li>Inoculation of few more colonies which grown on plate with transformation: <a href=http://partsregistry.org/Part:BBa_I739204>pACYC177</a> + <a href=http://partsregistry.org/Part:BBa_K103016>OmpA-linker-omega-linker (BBa_K103016)</a> to liquid LB + kanamycin. </li></ol>
 +
 
 +
<a name="fig2"><img src="https://static.igem.org/mediawiki/2008/0/0e/Traw_05_10_2008.jpg"></a>
 +
<var><b>Fig. 2.</b> Control digests of pACYC177+OmpA-linker-omega-linker (BBa_K103016)<br>
 +
1. Marker<br>
 +
2.-11.  Control digests of pACYC177+OmpA-linker-omega-linker (BBa_K103016)<br>
 +
12. Marker<br></var>
 +
 
 +
 
 +
<h3>Preparation of vector for pT7 constructs</h3>
<h4>Piotr</h4>
<h4>Piotr</h4>
-
<ol><li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#chemotransform">Transformation</a> of <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top10">TOP10</a> with ligations <a href=http://partsregistry.org/Part:pSB2K3>pSB2K3</a> + pLac_OmpA_omega (without EcoRI site) and <a hrefhttps://2008.igem.org/Wiki/Team:Warsaw/vectors/pET15b%2BOmpA-omega>pET15b+OmpA_omega</a> without XbaI.</li>
+
<ol><li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#chemotransform">Transformation</a> of <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top10">TOP10</a> with selfligation of <a hrefhttps://2008.igem.org/Wiki/Team:Warsaw/vectors/pET15b%2BOmpA-omega>pET15b+OmpA_omega</a> (with removed XbaI site).</li>
-
<li> Plating: <a href=http://partsregistry.org/Part:pSB2K3>pSB2K3</a> + pLac_OmpA_omega (without EcoRI site) on LB with kanamycin and pET15b+OmpA_omega without XbaI on LB with ampicillin.</li>
+
<li> Plating on LB with ampicillin.</li></ol>
-
<li>Inoculation of few more colonies which grown on plates with three separate transformations: <a href=http://partsregistry.org/Part:pSB2K3>pSB2K3</a> + _alpha, <a href=http://partsregistry.org/Part:pSB2K3>pSB2K3</a> + _omega and pACYC177 + OmpA_omega_ to liquid LB + kanamycin. </li>
+
 
 +
 
 +
 
 +
<h3>Preparation of <a href=http://partsregistry.org/Part:BBa_K103018>OmpA_linker_omega_linker under Plac (BBa_K103018)</a></h3>
 +
<h4>Piotr</h4>
 +
<ol><li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#chemotransform">Transformation</a> of <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top10">TOP10</a> with ligations <a href=http://partsregistry.org/Part:pSB2K3>pSB2K3</a> + <a href=http://partsregistry.org/Part:BBa_K103018>BBa_K103018</a> (without internal EcoRI site).</li>
 +
<li> Plating on LB with kanamycin.</li>
 +
 
 +
</ol>
 +
 
 +
<h3>Preparation of <a href=http://partsregistry.org/Part:BBa_K103001>AID(BBa_K103001)</a></h3>
 +
<h4>Michał K., Piotr</h4>
 +
<ol><li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#ligation">Ligation</a> of DNA fragments: <a href=http://partsregistry.org/wiki/index.php?title=Part:pSB1A3>pSB1A3</a>+ <a href=http://partsregistry.org/Part:BBa_K103001>AID(BBa_K103001)</a> (from <a href=https://2008.igem.org/Team:Warsaw/Calendar-Main/1_October_2008>1 October</a>).</li>
 +
<li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#chemotransform">Transformation</a> of <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top10">TOP10</a> with above ligation.</li>
 +
<li> Plating on LB with ampicillin.</li></ol>
 +
 
 +
 
 +
<h3>Preparation of <a href=http://partsregistry.org/Part:BBa_K103002>AID under pBAD/araC (BBa_K103002)</a></h3>
 +
<h4>Piotr</h4>
 +
<ol>
 +
 
<li><a href="https://2008.igem.org/Wiki/Team:Warsaw/protocols#digest">Digest</a> of <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pMPM-T5-AID>pMPMT5+AID</a> with EcoRI (EcoRI buffer). DNA ends <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#blunting">blunting</a> with Klenow fragment. </li>
<li><a href="https://2008.igem.org/Wiki/Team:Warsaw/protocols#digest">Digest</a> of <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pMPM-T5-AID>pMPMT5+AID</a> with EcoRI (EcoRI buffer). DNA ends <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#blunting">blunting</a> with Klenow fragment. </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/pMPM-T5-AID>pMPMT5+AID</a> without EcoRI site.</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/pMPM-T5-AID>pMPMT5+AID</a> to remove EcoRI site.</li>
</ol>
</ol>

Latest revision as of 21:22, 28 October 2008

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Preparation of linker_alpha (BBa_K103009)

Michał K., Piotr

  1. Isolation of plasmid from culture inoculated on previous day (pSB2K3 + linker_alpha (BBa_K103009)).
  2. Control digest of isolated plasmid with EcoRI and PstI (Orange buffer). Gel electrophoresis - no proper clones found. Fig. 1.
  3. Inoculation of few more colonies which grown on plate with transformation: pSB2K3 + linker_alpha (BBa_K103009) to liquid LB + kanamycin.

    4. Fig. 1.Control EcoRI/PstI digest of pSB2K3+linker_omega (BBa_K103013)
    1. and 12. Marker
    2-9. Control digests of pSB2K3+linker_alpha (BBa_K103009)
    10-17. Control digests of pSB2K3+linker_omega (BBa_K103013)

Preparation of linker_omega (BBa_K103013)

Michał K., Piotr

  1. Isolation of plasmid from culture inoculated on previous day (pSB2K3 + linker_omega (BBa_K103013)).
  2. Control digest of isolated plasmid with EcoRI and PstI (Orange buffer). Gel electrophoresis - no proper clones found. Fig. 1.
  3. Inoculation of few more colonies which grown on plates with transformation: pSB2K3 + linker_omega (BBa_K103013) to liquid LB + kanamycin.
Fig. 1.Control EcoRI/PstI digest of pSB2K3+linker_omega (BBa_K103013)
1. and 12. Marker
2-9. Control digests of pSB2K3+linker_alpha (BBa_K103009)
10-17. Control digests of pSB2K3+linker_omega (BBa_K103013)

Preparation of OmpA-linker-omega-linker (BBa_K103016)

Michał K., Piotr

  1. Isolation of plasmid from culture inoculated on previous day (pACYC177 + OmpA-linker-omega-linker (BBa_K103016)).
  2. Control digest of isolated plasmid with EcoRI and PstI (Orange buffer). Gel electrophoresis - no proper clones found. Fig. 2.
  3. Inoculation of few more colonies which grown on plate with transformation: pACYC177 + OmpA-linker-omega-linker (BBa_K103016) to liquid LB + kanamycin.
Fig. 2. Control digests of pACYC177+OmpA-linker-omega-linker (BBa_K103016)
1. Marker
2.-11. Control digests of pACYC177+OmpA-linker-omega-linker (BBa_K103016)
12. Marker

Preparation of vector for pT7 constructs

Piotr

  1. Transformation of TOP10 with selfligation of pET15b+OmpA_omega (with removed XbaI site).
  2. Plating on LB with ampicillin.

Preparation of OmpA_linker_omega_linker under Plac (BBa_K103018)

Piotr

  1. Transformation of TOP10 with ligations pSB2K3 + BBa_K103018 (without internal EcoRI site).
  2. Plating on LB with kanamycin.

Preparation of AID(BBa_K103001)

Michał K., Piotr

  1. Ligation of DNA fragments: pSB1A3+ AID(BBa_K103001) (from 1 October).
  2. Transformation of TOP10 with above ligation.
  3. Plating on LB with ampicillin.

Preparation of AID under pBAD/araC (BBa_K103002)

Piotr

  1. Digest of pMPMT5+AID with EcoRI (EcoRI buffer). DNA ends blunting with Klenow fragment.
  2. Overnight ligation of pMPMT5+AID to remove EcoRI site.

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