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Team:UCL/Project/Protocols - Revision history
2024-03-29T11:28:09Z
Revision history for this page on the wiki
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http://2013.igem.org/wiki/index.php?title=Team:UCL/Project/Protocols&diff=317350&oldid=prev
AlexBates at 03:58, 5 October 2013
2013-10-05T03:58:34Z
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><b><del class="diffchange diffchange-inline">9</del>)</b> Assay for gene expression. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><b><ins class="diffchange diffchange-inline">10</ins>)</b> Assay for gene expression. </div></td></tr>
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AlexBates
http://2013.igem.org/wiki/index.php?title=Team:UCL/Project/Protocols&diff=317216&oldid=prev
AlexBates at 03:55, 5 October 2013
2013-10-05T03:55:02Z
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><b>2)</b> Incubate cells in their normal growth conditions (37^0 C and 5% CO2) for 24 hours.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><b>2)</b> Incubate cells in their normal growth conditions (37^0 C and 5% CO2) for 24 hours.</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"></p></del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"><p class="body_text"></del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"><b>3)</b> Dilute 2µg of DNA dissolved in TE buffer (min conc. 0.1µg/µl) with serum, protein and antibiotic free medium (to avoid macromolecular interference with complex formation) to a total of 100µl. Mix.</del></div></td><td colspan="2"> </td></tr>
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<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"><p class="body_text"></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"><b>3)</b> Dilute 2µg of DNA dissolved in TE buffer (min conc. 0.1µg/µl) with serum, protein and antibiotic free medium (to avoid macromolecular interference with complex formation) to a total of 100µl. Mix.</ins></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><b>4)</b> Add 10µl of superfect (SF) reagent to the solution. Vortex for 10 seconds.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><b>4)</b> Add 10µl of superfect (SF) reagent to the solution. Vortex for 10 seconds.</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Dissolve Aβ in dimethyl sulfoxide (Me2SO, Sigma) to a concentration of 5mM. Dilute in MQ water to a final concentration of 25 μm immediately prior to use.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Dissolve Aβ in dimethyl sulfoxide (Me2SO, Sigma) to a concentration of 5mM. Dilute in MQ water to a final concentration of 25 μm immediately prior to use.</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"></p></del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"><p class="body_text"></del></div></td><td colspan="2"> </td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;">To prepare Aβ fibrils (fAβ), dilute 5 mm Aβ1-42 or Aβ1-40 in Me2SO in 10 mm HCl to 100 μm (for Aβ1-42) or 200 μm (for Aβ1-40), vortex for 30 s, and incubate at 37 °C for 5 days.</del></div></td><td colspan="2"> </td></tr>
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<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"><p class="body_text"></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">To prepare Aβ fibrils (fAβ), dilute 5 mm Aβ1-42 or Aβ1-40 in Me2SO in 10 mm HCl to 100 μm (for Aβ1-42) or 200 μm (for Aβ1-40), vortex for 30 s, and incubate at 37 °C for 5 days.</ins></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>a. Activate pro-MMP-9 with 1 mm p-aminophenylmercuric acetate at 37 °C for 24 h prior to use. This step is not necessary with <a href="http://parts.igem.org/Part:BBa_K1018001" target="_blank">(BBa_K1018000)</a>, as it contains the active form. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>a. Activate pro-MMP-9 with 1 mm p-aminophenylmercuric acetate at 37 °C for 24 h prior to use. This step is not necessary with <a href="http://parts.igem.org/Part:BBa_K1018001" target="_blank">(BBa_K1018000)</a>, as it contains the active form. </div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;"><b>4)</b> Add 5–10µL of protein solution (or transfected HeLa/microglia lysate mixed with degraded amyloid - remember to also include a control) to the cuvette. Incubate for 30 min at room temperature. A red precipitate may become visible. Pipette the solution up and down to mix the contents. Take a record of the spectrum between 400 and 700 nm.</del></div></td><td colspan="2"> </td></tr>
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<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"><p class="body_text"></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"><b>4)</b> Add 5–10µL of protein solution (or transfected HeLa/microglia lysate mixed with degraded amyloid - remember to also include a control) to the cuvette. Incubate for 30 min at room temperature. A red precipitate may become visible. Pipette the solution up and down to mix the contents. Take a record of the spectrum between 400 and 700 nm.</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></p></ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p class="body_text"></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p class="body_text"></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><b>5)</b> Subtract mathematically the Congo Red spectrum from the protein/lysate-Congo Red spectrum. A maximal spectral difference at 540nm is indicative of amyloid fibrils.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><b>5)</b> Subtract mathematically the Congo Red spectrum from the protein/lysate-Congo Red spectrum. A maximal spectral difference at 540nm is indicative of amyloid fibrils.</div></td></tr>
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AlexBates
http://2013.igem.org/wiki/index.php?title=Team:UCL/Project/Protocols&diff=317138&oldid=prev
AlexBates at 03:52, 5 October 2013
2013-10-05T03:52:40Z
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AlexBates
http://2013.igem.org/wiki/index.php?title=Team:UCL/Project/Protocols&diff=317098&oldid=prev
AlexBates at 03:52, 5 October 2013
2013-10-05T03:52:00Z
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AlexBates
http://2013.igem.org/wiki/index.php?title=Team:UCL/Project/Protocols&diff=316953&oldid=prev
AlexBates at 03:48, 5 October 2013
2013-10-05T03:48:03Z
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AlexBates
http://2013.igem.org/wiki/index.php?title=Team:UCL/Project/Protocols&diff=316843&oldid=prev
AlexBates at 03:44, 5 October 2013
2013-10-05T03:44:27Z
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AlexBates
http://2013.igem.org/wiki/index.php?title=Team:UCL/Project/Protocols&diff=316753&oldid=prev
AlexBates at 03:41, 5 October 2013
2013-10-05T03:41:34Z
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AlexBates
http://2013.igem.org/wiki/index.php?title=Team:UCL/Project/Protocols&diff=316665&oldid=prev
AlexBates at 03:38, 5 October 2013
2013-10-05T03:38:26Z
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AlexBates
http://2013.igem.org/wiki/index.php?title=Team:UCL/Project/Protocols&diff=316555&oldid=prev
AlexBates at 03:35, 5 October 2013
2013-10-05T03:35:32Z
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AlexBates
http://2013.igem.org/wiki/index.php?title=Team:UCL/Project/Protocols&diff=316498&oldid=prev
AlexBates at 03:33, 5 October 2013
2013-10-05T03:33:55Z
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</table>
AlexBates