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Team:UNITN-Trento/Notebook/Labposts/07/28 - Revision history
2024-03-29T00:21:07Z
Revision history for this page on the wiki
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Ggirelli at 10:38, 3 October 2013
2013-10-03T10:38:51Z
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>{</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>{"date" : "2013-07-<ins class="diffchange diffchange-inline">11</ins>","author" : "<ins class="diffchange diffchange-inline">gabriele</ins>","title" : "<ins class="diffchange diffchange-inline">Extraction </ins>(<ins class="diffchange diffchange-inline">again</ins>) <ins class="diffchange diffchange-inline">and ligation</ins>","content" : "<html><ins class="diffchange diffchange-inline"><h3>SAM synthetase extraction</h3></ins><a href=\"https://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/Notebook#tn-post-2013-07-<ins class="diffchange diffchange-inline">10</ins>-<ins class="diffchange diffchange-inline">gabriele-viola</ins>\"><ins class="diffchange diffchange-inline">Last time</ins></a> I <ins class="diffchange diffchange-inline">determined the correct protocol </ins>to <ins class="diffchange diffchange-inline">extract SAM synthetase from </ins>the <ins class="diffchange diffchange-inline"><i>E. coli</i> using the new plasmid (at the end</ins>, <ins class="diffchange diffchange-inline">the protocol was <</ins>a <ins class="diffchange diffchange-inline">href=\"https://2013</ins>.<ins class="diffchange diffchange-inline">igem</ins>.<ins class="diffchange diffchange-inline">org/Team:UNITN-Trento/Protocols#SAM-extraction-genome-coli\">the usual one</a>). I performed three PCRs (G1</ins>, <ins class="diffchange diffchange-inline">G2, G3) </ins>and <ins class="diffchange diffchange-inline">run the products on 1% agarose gel</ins>.</html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|<ins class="diffchange diffchange-inline">Gel</ins>|<html><center<ins class="diffchange diffchange-inline">><table class=\"tn-sp-table\"><tr><th colspan=\"4\">Loading scheme</th></tr><tr><td>1kb ladder</td><td>G1</td><td>G2</td><td>G3</td></tr></table</ins>><img src=\"https://static.igem.org/mediawiki/2013/<ins class="diffchange diffchange-inline">f</ins>/<ins class="diffchange diffchange-inline">f4</ins>/Tn-<ins class="diffchange diffchange-inline">20130711</ins>-<ins class="diffchange diffchange-inline">GG_SAMsynthetase_extraction_EXSP</ins>.<ins class="diffchange diffchange-inline">jpg</ins>\" <ins class="diffchange diffchange-inline">alt</ins>=<ins class="diffchange diffchange-inline">\"Gel\" title=\"Gel</ins>\" width<ins class="diffchange diffchange-inline">=\"</ins>450px\" <ins class="diffchange diffchange-inline">/</ins>></center></html>}}<html><ins class="diffchange diffchange-inline"><br><hr><h3>Ligation</h3>At first</ins>, <ins class="diffchange diffchange-inline">I added 1&micro;l of SAP to pSB1C3 O/N digestion and 1&micro;l of DpnI to SAMsynthetase O/N digestion, and incubated </ins>the <ins class="diffchange diffchange-inline">samples at 37&deg;C for 1</ins>.<ins class="diffchange diffchange-inline">5h. Then I quantified </ins>the <ins class="diffchange diffchange-inline">digestions</ins>.<ins class="diffchange diffchange-inline"><center><table class=\"tn-sp-table\"><tr><th>Sample</th><th>Quantity</th></tr><tr><td>SAM synthetase</td><td>12.2ng/&micro;l</td></tr><tr><td>pSB1C3</td><th>14.0ng/&micro;l</th></tr></table></center><br/>Then I performed the ligation and left the reaction run for 2h at room temperature.</html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Ligation Mixes|<html><center><table class=\"tn-sp-table\"><tr><td style=\"border:none;\"></td><th>CTRL</th><th>1:1</th><th>1:2</th></tr><tr><td>Buffer</td><td colspan=\"3\">4&micro;l</td></tr><tr><td>Plasmid</td><td colspan=\"3\">14.29&micro;l</td></tr><tr><td>Insert</td><td>0</td><td>9.15&micro;l</td><td>18.3&micro;l</td></tr><tr><td>Ligase</td><td colspan=\"3\">1&micro;l</td></tr><tr><td>Water</td><td>20.71&micro;l</td><td>11.56&micro;l</td><td>2.41&micro;l</td></tr></table></center></html>}}<html>Then I transformed the ligation products in NEB10&beta;</ins></html>","tags" : "<ins class="diffchange diffchange-inline">SAMsynthetase</ins>"}</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline"> </del>"date" : "2013-07-<del class="diffchange diffchange-inline">15</del>",</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline"> </del>"author" : "<del class="diffchange diffchange-inline">thomas</del>",</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline"> </del>"title" : "<del class="diffchange diffchange-inline">Ethylene production kinetic assay </del>(<del class="diffchange diffchange-inline">third and last trial</del>)",</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline"> </del>"content" : "<html><del class="diffchange diffchange-inline">Since I was not satisfied with the curve obtained during the </del><a href=\"https://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/Notebook#tn-post-2013-07-<del class="diffchange diffchange-inline">08</del>-<del class="diffchange diffchange-inline">thomas</del>\"><del class="diffchange diffchange-inline">last experiment</del></a><del class="diffchange diffchange-inline">, </del>I <del class="diffchange diffchange-inline">decided </del>to <del class="diffchange diffchange-inline">redo </del>the <del class="diffchange diffchange-inline">experiment</del>, <del class="diffchange diffchange-inline">this time starting from </del>a <del class="diffchange diffchange-inline">culture with O</del>.<del class="diffchange diffchange-inline">D</del>.<del class="diffchange diffchange-inline">600 = 0</del>,<del class="diffchange diffchange-inline">8 </del>and <del class="diffchange diffchange-inline">registering a measurment every 45 min</del>. <del class="diffchange diffchange-inline"> <br/></del></html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|<del class="diffchange diffchange-inline">Plot</del>|<html><center><img src=\"https://static.igem.org/mediawiki/2013/<del class="diffchange diffchange-inline">0</del>/<del class="diffchange diffchange-inline">00</del>/Tn-<del class="diffchange diffchange-inline">2013_kinetic_EFE_plot</del>-<del class="diffchange diffchange-inline">2</del>.<del class="diffchange diffchange-inline">png</del>\" <del class="diffchange diffchange-inline">style </del>=\"width<del class="diffchange diffchange-inline">: </del>450px\"></center></html>}}<html><del class="diffchange diffchange-inline">Interestingly</del>, <del class="diffchange diffchange-inline">inducing with arabinose when </del>the <del class="diffchange diffchange-inline">sample has reached OD600 0</del>.<del class="diffchange diffchange-inline">8 causes an increase of more than two fold in </del>the <del class="diffchange diffchange-inline">production of ethylene</del>.</html>",</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline"> </del>"tags" : "<del class="diffchange diffchange-inline">EFE</del>"</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>}</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></div></td></tr>
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Ggirelli
http://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/Notebook/Labposts/07/28&diff=273366&oldid=prev
Ggirelli at 08:32, 3 October 2013
2013-10-03T08:32:16Z
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<td colspan='2' style="background-color: white; color:black;">← Older revision</td>
<td colspan='2' style="background-color: white; color:black;">Revision as of 08:32, 3 October 2013</td>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> "date" : "2013-07-<del class="diffchange diffchange-inline">30</del>",</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> "date" : "2013-07-<ins class="diffchange diffchange-inline">15</ins>",</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> "author" : "thomas",</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> "author" : "thomas",</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> "title" : "<del class="diffchange diffchange-inline">Bacillus transformation!!!</del>",</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> "title" : "<ins class="diffchange diffchange-inline">Ethylene production kinetic assay (third and last trial)</ins>",</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> "content" : "<html><del class="diffchange diffchange-inline">In order to test </del>the <del class="diffchange diffchange-inline">transformation protocol for <i>B. subtilis</i> that Groeningen team kindly sent to us I prepared a competent cell Media following </del><a href=\"https://2013.igem.org/Team:UNITN-Trento/<del class="diffchange diffchange-inline">Protocols</del>#<del class="diffchange diffchange-inline">subtilis</del>-<del class="diffchange diffchange-inline">transformation</del>\"><del class="diffchange diffchange-inline">this protocol</del></a><del class="diffchange diffchange-inline">. </del>I <del class="diffchange diffchange-inline">proceeded then perfonming the transformation using </del>the <del class="diffchange diffchange-inline">empty vector BBa_K823026. I finally plated the tranformed cells into LB agar plates </del>with <del class="diffchange diffchange-inline">10 ug/ml of Kanamycin</del>. <del class="diffchange diffchange-inline">The day after took a picture of the plate </del>and <del class="diffchange diffchange-inline">performed </del>a <del class="diffchange diffchange-inline">Gram staining following <a href=\"\">this protocol</del></<del class="diffchange diffchange-inline">a</del>><del class="diffchange diffchange-inline">. </del></html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|<del class="diffchange diffchange-inline">Plate image</del>|<html><center><img src=\"https://static.igem.org/mediawiki/2013/<del class="diffchange diffchange-inline">e</del>/<del class="diffchange diffchange-inline">ec</del>/Tn-<del class="diffchange diffchange-inline">2013_bacillus_transformation_plate_1</del>.<del class="diffchange diffchange-inline">jpg</del>\" style =\"width: 450px\"></center></html>}}<del class="diffchange diffchange-inline">{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Bacillus microscope image|</del><html><del class="diffchange diffchange-inline"><center><img src=\"https://static</del>.<del class="diffchange diffchange-inline">igem</del>.<del class="diffchange diffchange-inline">org/mediawiki/2013/6/65/Tn-2013_gram_staining_bacillus.jpg\" style =\"width: 450px\"></center></html>}}<html> As you can see from the pictures, seems that <i>Bacillus</i> was successful transformed!!!!</del></html>",</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> "content" : "<html><ins class="diffchange diffchange-inline">Since I was not satisfied with the curve obtained during </ins>the <a href=\"https://2013.igem.org/<ins class="diffchange diffchange-inline">wiki/index.php?title=</ins>Team:UNITN-Trento/<ins class="diffchange diffchange-inline">Notebook</ins>#<ins class="diffchange diffchange-inline">tn</ins>-<ins class="diffchange diffchange-inline">post-2013-07-08-thomas</ins>\"><ins class="diffchange diffchange-inline">last experiment</ins></a><ins class="diffchange diffchange-inline">, </ins>I <ins class="diffchange diffchange-inline">decided to redo </ins>the <ins class="diffchange diffchange-inline">experiment, this time starting from a culture </ins>with <ins class="diffchange diffchange-inline">O.D</ins>.<ins class="diffchange diffchange-inline">600 = 0,8 </ins>and <ins class="diffchange diffchange-inline">registering </ins>a <ins class="diffchange diffchange-inline">measurment every 45 min. </ins><<ins class="diffchange diffchange-inline">br</ins>/></html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|<ins class="diffchange diffchange-inline">Plot</ins>|<html><center><img src=\"https://static.igem.org/mediawiki/2013/<ins class="diffchange diffchange-inline">0</ins>/<ins class="diffchange diffchange-inline">00</ins>/Tn-<ins class="diffchange diffchange-inline">2013_kinetic_EFE_plot-2</ins>.<ins class="diffchange diffchange-inline">png</ins>\" style =\"width: 450px\"></center></html>}}<html><ins class="diffchange diffchange-inline">Interestingly, inducing with arabinose when the sample has reached OD600 0</ins>.<ins class="diffchange diffchange-inline">8 causes an increase of more than two fold in the production of ethylene</ins>.</html>",</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> "tags" : "<del class="diffchange diffchange-inline">b. subtilis-pspac</del>"</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> "tags" : "<ins class="diffchange diffchange-inline">EFE</ins>"</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>}</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>}</div></td></tr>
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Ggirelli
http://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/Notebook/Labposts/07/28&diff=110192&oldid=prev
Ggirelli: moved Team:UNITN-Trento/Notebook/Labposts/127 to Team:UNITN-Trento/Notebook/Labposts/07/28
2013-09-09T07:27:31Z
<p>moved <a href="/Team:UNITN-Trento/Notebook/Labposts/127" class="mw-redirect" title="Team:UNITN-Trento/Notebook/Labposts/127">Team:UNITN-Trento/Notebook/Labposts/127</a> to <a href="/Team:UNITN-Trento/Notebook/Labposts/07/28" title="Team:UNITN-Trento/Notebook/Labposts/07/28">Team:UNITN-Trento/Notebook/Labposts/07/28</a></p>
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<td colspan='1' style="background-color: white; color:black;">← Older revision</td>
<td colspan='1' style="background-color: white; color:black;">Revision as of 07:27, 9 September 2013</td>
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Ggirelli
http://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/Notebook/Labposts/07/28&diff=78628&oldid=prev
Ggirelli: Created page with "{ "date" : "2013-07-30", "author" : "thomas", "title" : "Bacillus transformation!!!", "content" : "<html>In order to test the transformation protocol for <i>B. subtilis</i> t..."
2013-08-19T13:45:47Z
<p>Created page with "{ "date" : "2013-07-30", "author" : "thomas", "title" : "Bacillus transformation!!!", "content" : "<html>In order to test the transformation protocol for <i>B. subtilis</i> t..."</p>
<p><b>New page</b></p><div>{<br />
"date" : "2013-07-30",<br />
"author" : "thomas",<br />
"title" : "Bacillus transformation!!!",<br />
"content" : "<html>In order to test the transformation protocol for <i>B. subtilis</i> that Groeningen team kindly sent to us I prepared a competent cell Media following <a href=\"https://2013.igem.org/Team:UNITN-Trento/Protocols#subtilis-transformation\">this protocol</a>. I proceeded then perfonming the transformation using the empty vector BBa_K823026. I finally plated the tranformed cells into LB agar plates with 10 ug/ml of Kanamycin. The day after took a picture of the plate and performed a Gram staining following <a href=\"\">this protocol</a>. </html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Plate image|<html><center><img src=\"https://static.igem.org/mediawiki/2013/e/ec/Tn-2013_bacillus_transformation_plate_1.jpg\" style =\"width: 450px\"></center></html>}}{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Bacillus microscope image|<html><center><img src=\"https://static.igem.org/mediawiki/2013/6/65/Tn-2013_gram_staining_bacillus.jpg\" style =\"width: 450px\"></center></html>}}<html> As you can see from the pictures, seems that <i>Bacillus</i> was successful transformed!!!!</html>",<br />
"tags" : "b. subtilis-pspac"<br />
}</div>
Ggirelli