Team:Warsaw/Calendar-Main/20 June 2008

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<h3>Preparation of constructs: OmpA_alpha and OmpA_omega</h3>
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<h4>Michał K.</h4>
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<ol>
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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 plasmids from cultures inoculated on previous day.</li>
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<li>Control <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest">digest</a> of isolated plasmids with PstI (Orange buffer).</li>
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<li>Gel electrophoresis (<a href="https://2008.igem.org/wiki/index.php?title=Team:Warsaw/Calendar-Main/20_June_2008#fig1">Fig. 1 and 2)</a>, no proper colonies found.</li></ol>
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<a name="fig1"><img src="https://static.igem.org/mediawiki/2008/7/78/Omp_control_digest_WAW.jpg" width=400/></a>
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<var><b>Fig. 1.</b> Lanes 1 to 8 - control digest of 8 clones carrying hypothetical OmpA_alpha fusion with PstI.</var>
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<img src="https://static.igem.org/mediawiki/2008/1/1a/Omp_control_digest2_WAW.jpg" width=400/>
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<var><b>Fig. 2.</b> Lanes 1 to 8 - control digest of 8 clones carrying hypothetical OmpA_omega fusion with PstI.</var>
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<h3>Preparation of alpha-A and omega-A fusions</h3>
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<h4>Michał L., Ewa, Marcin</h4>
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<table id="result">
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<tr ><th colspan="4"><a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a></td></tr>
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<tr><th>Product</th><th>Template</th><th>Primers</th><th>Product length</th></tr>
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<tr><th>linker-A</th><td>pDRIVE-TapTag</td><td><a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AL+link10+homo2">AL+link10+homo2</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AP+NotI">AP+NotI</a> </td><td>470 bp</td></tr>
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<tr><th>alpha-linker</th><td><a href=http://www.fermentas.com/techinfo/nucleicacids/mappuc1819.htm>pUC19</a></td><td> <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AlphaL+SacI">AlphaL+SacI</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#AlphaP+link10+homo2">AlphaP+link10+homo2</a></td><td>600 bp</td></tr>
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<tr><th>omega-linker</th><td><a href=http://www.fermentas.com/techinfo/nucleicacids/mappuc1819.htm>pUC19</a></td><td><a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#OmegaL+SacI">OmegaL+SacI</a> and <a href="https://2008.igem.org/Wiki/Team:Warsaw/primers#OmegaP+link10+homo2">OmegaP+link10+homo2</a></td><td>400 bp</td></tr>
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</table>
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<br><br><br>
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<table id="result">
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<b>Universal PCR program for protein A, alpha and omega</b><br>
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<tr><th>Temperature</th><th>Time</th></tr>
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<tr><td>94&deg;C</td><td>4:00</td></tr>
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<tr><td>94&deg;C</td><td>0:30</td><td rowspan="3">28 cycles</td></tr>
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<tr><td>50&deg;C</td><td>0:45</td></tr>
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<tr><td>72&deg;C</td><td>2:00</td></tr>
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<tr><td>72&deg;C</td><td>10:00</td></tr>
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<tr><td>4&deg;C</td><td>infinite</td></tr>
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</table>
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<br>
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</html>

Latest revision as of 14:58, 26 October 2008

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Preparation of constructs: OmpA_alpha and OmpA_omega

Michał K.

  1. Isolation of plasmids from cultures inoculated on previous day.
  2. Control digest of isolated plasmids with PstI (Orange buffer).
  3. Gel electrophoresis (Fig. 1 and 2), no proper colonies found.
Fig. 1. Lanes 1 to 8 - control digest of 8 clones carrying hypothetical OmpA_alpha fusion with PstI. Fig. 2. Lanes 1 to 8 - control digest of 8 clones carrying hypothetical OmpA_omega fusion with PstI.

Preparation of alpha-A and omega-A fusions

Michał L., Ewa, Marcin

PCR
ProductTemplatePrimersProduct length
linker-ApDRIVE-TapTagAL+link10+homo2 and AP+NotI 470 bp
alpha-linkerpUC19 AlphaL+SacI and AlphaP+link10+homo2600 bp
omega-linkerpUC19OmegaL+SacI and OmegaP+link10+homo2400 bp



Universal PCR program for protein A, alpha and omega
TemperatureTime
94°C4:00
94°C0:3028 cycles
50°C0:45
72°C2:00
72°C10:00
4°Cinfinite

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