Team:Warsaw/Calendar-Main/11 July 2008

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<h3>Preparation of constructs with OmpA protein fusions</h3>
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<h3>Preparation of construct <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pKSII+A>pKS with A protein</a></h3>
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<h4>Michał L., Ewa, Marcin</h4>
<p><ol>
<p><ol>
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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 OmpA_alpha. </li>
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<li> Colony PCR on colonies from plates with transformations <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pKSII+A>pKS-A</a>. <br>
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<li> Confirmed transformant colonies inoculated to liquid LB with kanamycin.</li>
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Primers used:
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</ol></p>
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<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AL+SacI">AL+SacI</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AP+NotI">AP+NotI</a>
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</li><li> Gel electrophoresis.</li>
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<li> Confirmed transformant colonies inoculated to liquid LB with ampicillin.</li></ol></p>
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<h3>Cloning omega-A fusion on pKS (second attempt)</h3>
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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> in place of OmpA</h3>
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<h4>Michał L., Ewa, Marcin:</h4>
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<h4>Piotr, Antoni</h4>
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<p><a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#plasmid_DNA_isolation>Isolation</a> of plasmids from cultures inoculated on previous day.</p>
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<p>We had to start form scratch with this one.
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<h3>Preparation of constructs: OmpA_alpha and OmpA_omega #2</h3>
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<ol>
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<li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> A in 50 µl<br>
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template DNA - pKS-A4 1 µl<br>
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primer <html>
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<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AP+NotI_N">AP+NotI_N</a> - 2 µl<br>
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primer
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<a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AL+link10+homo2_N">AL+link10+homo2_N</a> - 2 µl<br>
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Pfu polymerase buffer + Mg<sup>2+</sup> (from Fermentas) - 5 µl<br>
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dNTPs - 1 µl <br>
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Pfu turbo - 0.5 µl<br>
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H2o - 38.5 µl<br>
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<br>
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Program:<br>
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<ol>
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<li> 95&deg;C 3'</li>
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<li> 95&deg;C 30"</li>
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<li>62&deg;C 45"</li>
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<li>72&deg;C 45"</li>
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<li>72&deg;C 10'</li>
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<li>keeping in 4&deg;C</li></ol>
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</li>
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<li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> omega in 50 µl<br>
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template DNA - pUC19 1 µl<br>
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primer OmegaLS - 2 µl<br>
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primer AOmegaPli - 2 µl<br>
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Pfu polymerase buffer + Mg<sup>2+</sup> (from Fermentas) - 5 µl<br>
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dNTPs - 1 µl <br>
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Pfu turbo - 0.5 µl<br>
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H2o - 38.5 µl<br>
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Program:<br>
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<h4>Michał K.</h4>
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<ol>
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<li> 95&deg;C 3'</li>
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<li> 95&deg;C 30"</li>
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<li> 62&deg;C 45"</li>
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<li> 72&deg;C 45"</li>
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<li> 72&deg;C 10'</li>
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<li> keeping in 4&deg;C</li></ol>
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25 cycles
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</li>
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<li> Gel electrophoresis</li>
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<li><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#DNA_isolation_from_agarose_gel">Reisolation</a> from agarose gel</li>
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</ol>
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</p>
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<h3>Preparation of construct pKS with A protein</h3>
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<p><ol>
<p><ol>
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<li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#plasmid_DNA_isolation">Isolation of plasmid DNA</a> from cultures inocluated on previous day. </li>
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<li> Colony <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> with <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#OmpaL_N">OmpaL_N</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#OmpaP_link">OmpaP_link</a>
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<li> Control <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">digest</a> of isolated plasmid with SacI and NotI; 2 h</li>
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<li> Gel electrophoresis of digested DNA</li>
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<li> We are glad to find the proper clone ;-) and inoculate liquid LB with ampicillin to freeze bacteria carrying pKS-A construct</li></ol>
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</p>
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primers on colonies from plates with transformations OmpA_alpha (annealing temperature - 55&deg;C,45 s of elongation step). </li>
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<li> Gel electrophoresis (<a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/11_July_2008#fig1">Fig. 1.</a>).</li>
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<li> Confirmed transformant colonies inoculated to liquid LB with kanamycin.</li>
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</ol></p>
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<a name="fig1"><img src="https://static.igem.org/mediawiki/2008/7/7e/11_th_july.jpg" width=300 /></a><var><b>Fig. 1. </b>Colony PCR using: OmpaL_N and OmpaP_link primers<b></b><br>
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Upper<br>
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1. Marker<br>
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2-13. colony PCR products carrying probable OmpA-alpha<br>
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Lower<br>
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1. Marker<br>
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2-13. colony PCR products carrying probable OmpA-alpha<br></var>

Latest revision as of 17:44, 26 October 2008

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Preparation of construct pKS with A protein

Michał L., Ewa, Marcin

  1. Colony PCR on colonies from plates with transformations pKS-A.
    Primers used: AL+SacI and AP+NotI
  2. Gel electrophoresis.
  3. Confirmed transformant colonies inoculated to liquid LB with ampicillin.

Cloning of protein Z DNA to pET15b-OmpA-alpha in place of OmpA

Piotr, Antoni

Isolation of plasmids from cultures inoculated on previous day.

Preparation of constructs: OmpA_alpha and OmpA_omega #2

Michał K.

  1. Colony PCR with OmpaL_N and OmpaP_link primers on colonies from plates with transformations OmpA_alpha (annealing temperature - 55°C,45 s of elongation step).
  2. Gel electrophoresis (Fig. 1.).
  3. Confirmed transformant colonies inoculated to liquid LB with kanamycin.

Fig. 1. Colony PCR using: OmpaL_N and OmpaP_link primers
Upper
1. Marker
2-13. colony PCR products carrying probable OmpA-alpha
Lower
1. Marker
2-13. colony PCR products carrying probable OmpA-alpha

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