http://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/Notebook/Labposts/07/36&feed=atom&action=historyTeam:UNITN-Trento/Notebook/Labposts/07/36 - Revision history2024-03-29T01:31:56ZRevision history for this page on the wikiMediaWiki 1.16.5http://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/Notebook/Labposts/07/36&diff=274456&oldid=prevGgirelli at 10:40, 3 October 20132013-10-03T10:40:40Z<p></p>
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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">17</ins>","author" : "<ins class="diffchange diffchange-inline">gabriele-</ins>caterina","title" : "<ins class="diffchange diffchange-inline">A short day</ins>","content" : "<html><ins class="diffchange diffchange-inline"><h3>Screening with AgeI</h3>First</ins>, <ins class="diffchange diffchange-inline">Caterina </ins>performed <ins class="diffchange diffchange-inline">a screening using AgeI on the \"1:1 A\" sample </ins>from <ins class="diffchange diffchange-inline"><a href=\"https://2013</ins>.<ins class="diffchange diffchange-inline">igem.org/wiki/index.php?title=Team:UNITN-Trento/Notebook#tn-post-2013-07-15-gabriele\">15/07</a>.</html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Digestion mix|<html><center><table class=\"tn-sp-table\"><tr><td style=\"border:none;\"></td><th>11A</th></tr><tr><td>template</td><td>4.34&micro;l</td></tr><tr><td>AgeI-HF</td><td rowspan=\"2\">1&micro;l</td></tr><tr><td>EcoRI-HF</td></tr><tr><td>NEBuffer4</td><td rowspan=\"2\">2&micro;l</td></tr><tr><td>BAS</td></tr><tr><td>Water</td><td>9.66&micro;l</td></tr></table></center></html>}}<html>But then </ins>we <ins class="diffchange diffchange-inline">realized </ins>the <ins class="diffchange diffchange-inline">Gabriele </ins>was <ins class="diffchange diffchange-inline">mistaken, AgeI is able to cut both Plac+</ins>RFP and <ins class="diffchange diffchange-inline">SAMsynthetase so it is </ins>not <ins class="diffchange diffchange-inline">the correct enzyme to perform the screening with...<br><hr><h3>Short digestion</h3>Gabriele then performed a short digestion (aka: a digestion similar to the screening run for 2 hours) with the sample G2_EX-SAMsynth-SP (62.6ng/&micro;l from <a href=\"https://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/Notebook#tn-post-2013-07-15-gabriele\">15/07</a>) and G4A_linear-pSB1C3 (41.9ng/&micro;l from <a href=\"https://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/Notebook#tn-post-2013-07-01-Gabriele\">01/07</a>).</html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Digestion mixes|<html><center><table class=\"tn-sp-table\"><tr><td style=\"border:none;\"></td><th>EX-</ins>SAM<ins class="diffchange diffchange-inline">-SP</th><th>linear-pSB1C3</th></tr><tr><td>template</td><td>24&micro;l</td><td>23</ins>.<ins class="diffchange diffchange-inline">87&micro;l</td></tr><tr><td>EcoRI-HF</td><td rowspan=\"2\" colspan=\"2\">1&micro;l</td></tr><tr><td>PstI</td></tr><tr><td>NEBuffer2</td><td rowspan=\"2\" colspan=\"2\">3&micro;l</td></tr><tr><td>BSA</td></tr><tr><td>water</td><td>3&micro;l</td><td>3.13&micro;l</td></tr></table></center></html>}}<html>After </ins>the <ins class="diffchange diffchange-inline">2 hours of incubation, Gabriele added 1&micro;l of DpnI to the G2 sample and 1&micro;l of SAP to the G4A sample and incubated them at 37&deg;C for 1h</ins>.<br<ins class="diffchange diffchange-inline">><hr><h3>Digestions purification<</ins>/<ins class="diffchange diffchange-inline">h3>After that, Gabriele purificated both today's short digestion and the O/N digestion from <a href=\"https://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/Notebook#tn-post-2013-07-17-gabriele-caterina\">yesterday</a>.<center><table class=\"tn-sp-table\"><tr><th>Sample</th><th>Digestion</th><th>Quantity</th></tr><tr><td>EX-SAMsynth-SP</td><td>Short</td><td>39.0ng/&micro;l</td></tr><tr><td>linear-pSB1C3</td><td>Short</td><td>12.1ng/&micro;l</td></tr><tr><td>EX-SAMsynth-SP</td><td>O/N</td><td>13.1ng/&micro;l</td></tr><tr><td>linear-pSB1C3</td><td>O/N</td><td>7.8ng/&micro;l</td></tr></table></center</ins>><br<ins class="diffchange diffchange-inline">><hr><h3>Short digest Ligation<</ins>/<ins class="diffchange diffchange-inline">h3</ins>><ins class="diffchange diffchange-inline">So, Gabriele </ins>performed <ins class="diffchange diffchange-inline">a ligation of </ins>the <ins class="diffchange diffchange-inline">short digested samples.<center><table class=\"tn-sp-table\"><tr><td style=\"border:none;\"></td><th>Ctrl</th><th>1:1</th><th>1:2</th><th>1:3</th></tr><tr><td>buffer</td><td colspan=\"4\">3.5&micro;l</td></tr><tr><td>plasmid</td><td colspan=\"4\">5&micro;l</td></tr><tr><td>insert</td><td>0</td><td>8&micro;l</td><td>16&micro;l</td><td>26&micro;l</td></tr><tr><td>Ligase</td><td colspan=\"4\">1&micro;l</td></tr><tr><td>Water</td><td>25.5&micro;l</td><td>17.5&micro;l</td><td>9.5&micro;l</td><td>0</td></tr></table></center>Then the ligation mixes were incubated at room temperature for 2 hours.<br><hr><h3>O/N digest Ligation</h3>Finally, Gabriele performed a ligation </ins>of the <ins class="diffchange diffchange-inline">overnight digested samples.<center><table class=\"tn-sp-table\"><tr><td style=\"border:none;\"></td><th>Ctrl</th><th>1:1</th></tr><tr><td>Buffer</td><td colspan=\"2\">3.5&micro;l</td></tr><tr><td>Plasmid</td><td colspan=\"2\">15&micro;l</td></tr><tr><td>Insert</td><td>0</td><td>15&micro;l</td></tr><tr><td>Ligase</td><td colspan=\"2\">1&micro;l</td></tr><tr><td>Water</td><td>15.5&micro;l</td><td>0.5&micro;l</td></tr></table></center>Then, the ligation mixes were incubated at room temperature for 2 hourse</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">19</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" : "caterina",</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">SAM sythetase and other miniprep</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">Yesterday</del>, <del class="diffchange diffchange-inline">I </del>performed <del class="diffchange diffchange-inline">inocula </del>from <del class="diffchange diffchange-inline">different plates prepared by Gire</del>. <del class="diffchange diffchange-inline">After miniprep and screening </del>we <del class="diffchange diffchange-inline">had </del>the <del class="diffchange diffchange-inline">confirmation that into pSB1C3 there </del>was RFP and not SAM <del class="diffchange diffchange-inline">synthetase</del>. <del class="diffchange diffchange-inline">So on Monday we will repeat </del>the <del class="diffchange diffchange-inline">experiment</del>.<br/><br/><del class="diffchange diffchange-inline">Then I </del>performed the <del class="diffchange diffchange-inline">miniprep </del>of <del class="diffchange diffchange-inline">some inocula that I prepared </del>the <del class="diffchange diffchange-inline">yesterday containing K1065101 and K1065102</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">SAM-K1065101-K1065102</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>
</table>Ggirellihttp://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/Notebook/Labposts/07/36&diff=273384&oldid=prevGgirelli at 08:34, 3 October 20132013-10-03T08:34:39Z<p></p>
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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">02</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">19</ins>",</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> "author" : "<del class="diffchange diffchange-inline">emil</del>",</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> "author" : "<ins class="diffchange diffchange-inline">caterina</ins>",</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">Purification of the digestion products (K823026, K823024, E0840) ligation of the GFP in the 2 backbones </del>and <del class="diffchange diffchange-inline">transformation of the ligation products </del>",</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> "title" : "<ins class="diffchange diffchange-inline">SAM sythetase </ins>and <ins class="diffchange diffchange-inline">other miniprep</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">This morning </del>I <del class="diffchange diffchange-inline">added 1&micro;l of DPN1 to the GFP ligation and 1&micro;l of SAP(alkaline phosphatase) to the 2 backbones</del>.After <del class="diffchange diffchange-inline">1h 30 min I have stopped </del>the <del class="diffchange diffchange-inline">reaction by putting the reaction tubes at 80 C&deg;</del>.<del class="diffchange diffchange-inline">Afterwards I have purified </del>the <del class="diffchange diffchange-inline">products following the <a href=\"https://2013</del>.<del class="diffchange diffchange-inline">igem.org/Team:UNITN-Trento/Protocols#Promega-PCR-Gel\"> purification protocol</del></<del class="diffchange diffchange-inline">a</del>><del class="diffchange diffchange-inline">.Then I quantified the products with the following results:</del></<del class="diffchange diffchange-inline">html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Quantification|<html><table> <tr><th>Sample</th><th>Quantification</th></tr><tr><td>BBa_E0840</td><td> 15.8ng/&micro;l</td></tr><tr><td>BBa_K823026</td><td> 37.6ng/&micro;l</td></tr><tr><td>BBa_K823024</td><td> 36.4ng/&micro;l</td></tr></table></html>}}<html</del>>Then I performed the <del class="diffchange diffchange-inline">ligation(1:1,1:3,CTRL) </del>of <del class="diffchange diffchange-inline">the GFP with the 2 backbone individually and following the <a href=\"https://2013.igem.org/Team:UNITN-Trento/Protocols#Ligation\"> ligation protocol</a>.Finally </del>I <del class="diffchange diffchange-inline">transformed 10&micro;l of </del>the <del class="diffchange diffchange-inline">ligation protocol in Neb10&beta; </del>and <del class="diffchange diffchange-inline">plate them on ampicillin LB agar following the <a href=\"https://2013.igem.org/Team:UNITN-Trento/Protocols#Competent-cells-transformation\"> transformation protocol</a></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">Yesterday, </ins>I <ins class="diffchange diffchange-inline">performed inocula from different plates prepared by Gire</ins>. After <ins class="diffchange diffchange-inline">miniprep and screening we had </ins>the <ins class="diffchange diffchange-inline">confirmation that into pSB1C3 there was RFP and not SAM synthetase</ins>. <ins class="diffchange diffchange-inline">So on Monday we will repeat </ins>the <ins class="diffchange diffchange-inline">experiment</ins>.<<ins class="diffchange diffchange-inline">br</ins>/><<ins class="diffchange diffchange-inline">br</ins>/>Then I performed the <ins class="diffchange diffchange-inline">miniprep </ins>of <ins class="diffchange diffchange-inline">some inocula that </ins>I <ins class="diffchange diffchange-inline">prepared </ins>the <ins class="diffchange diffchange-inline">yesterday containing K1065101 </ins>and <ins class="diffchange diffchange-inline">K1065102</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">E0840</del>-<del class="diffchange diffchange-inline">K823026</del>-<del class="diffchange diffchange-inline">K823024</del>"</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> "tags" : "<ins class="diffchange diffchange-inline">SAM</ins>-<ins class="diffchange diffchange-inline">K1065101</ins>-<ins class="diffchange diffchange-inline">K1065102</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>
</table>Ggirellihttp://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/Notebook/Labposts/07/36&diff=110413&oldid=prevGgirelli: moved Team:UNITN-Trento/Notebook/Labposts/62 to Team:UNITN-Trento/Notebook/Labposts/07/362013-09-09T07:47:56Z<p>moved <a href="/Team:UNITN-Trento/Notebook/Labposts/62" class="mw-redirect" title="Team:UNITN-Trento/Notebook/Labposts/62">Team:UNITN-Trento/Notebook/Labposts/62</a> to <a href="/Team:UNITN-Trento/Notebook/Labposts/07/36" title="Team:UNITN-Trento/Notebook/Labposts/07/36">Team:UNITN-Trento/Notebook/Labposts/07/36</a></p>
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<td colspan='1' style="background-color: white; color:black;">Revision as of 07:47, 9 September 2013</td>
</tr></table>Ggirellihttp://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/Notebook/Labposts/07/36&diff=78524&oldid=prevGgirelli: Created page with "{ "date" : "2013-07-02", "author" : "emil", "title" : "Purification of the digestion products (K823026, K823024, E0840) ligation of the GFP in the 2 backbones and transformati..."2013-08-19T13:27:55Z<p>Created page with "{ "date" : "2013-07-02", "author" : "emil", "title" : "Purification of the digestion products (K823026, K823024, E0840) ligation of the GFP in the 2 backbones and transformati..."</p>
<p><b>New page</b></p><div>{<br />
"date" : "2013-07-02",<br />
"author" : "emil",<br />
"title" : "Purification of the digestion products (K823026, K823024, E0840) ligation of the GFP in the 2 backbones and transformation of the ligation products ",<br />
"content" : "<html>This morning I added 1&micro;l of DPN1 to the GFP ligation and 1&micro;l of SAP(alkaline phosphatase) to the 2 backbones.After 1h 30 min I have stopped the reaction by putting the reaction tubes at 80 C&deg;.Afterwards I have purified the products following the <a href=\"https://2013.igem.org/Team:UNITN-Trento/Protocols#Promega-PCR-Gel\"> purification protocol</a>.Then I quantified the products with the following results:</html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Quantification|<html><table> <tr><th>Sample</th><th>Quantification</th></tr><tr><td>BBa_E0840</td><td> 15.8ng/&micro;l</td></tr><tr><td>BBa_K823026</td><td> 37.6ng/&micro;l</td></tr><tr><td>BBa_K823024</td><td> 36.4ng/&micro;l</td></tr></table></html>}}<html>Then I performed the ligation(1:1,1:3,CTRL) of the GFP with the 2 backbone individually and following the <a href=\"https://2013.igem.org/Team:UNITN-Trento/Protocols#Ligation\"> ligation protocol</a>.Finally I transformed 10&micro;l of the ligation protocol in Neb10&beta; and plate them on ampicillin LB agar following the <a href=\"https://2013.igem.org/Team:UNITN-Trento/Protocols#Competent-cells-transformation\"> transformation protocol</a>.</html>",<br />
"tags" : "E0840-K823026-K823024"<br />
}</div>Ggirelli