http://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/Notebook/Labposts/07/25&feed=atom&action=historyTeam:UNITN-Trento/Notebook/Labposts/07/25 - Revision history2024-03-28T22:47:39ZRevision history for this page on the wikiMediaWiki 1.16.5http://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/Notebook/Labposts/07/25&diff=274414&oldid=prevGgirelli at 10:38, 3 October 20132013-10-03T10:38:11Z<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">10</ins>","author" : "gabriele<ins class="diffchange diffchange-inline">-viola</ins>","title" : "<ins class="diffchange diffchange-inline">Reboot: starting from the beginning</ins>","content" : "<html><ins class="diffchange diffchange-inline">Since </ins>the <ins class="diffchange diffchange-inline">new forward primer (with the entire prefix) arrived, I started again </ins>from <ins class="diffchange diffchange-inline">the beginning.<br/><br/><h3>SAM synthetase extraction</h3>The new primer forward has just the EcoRI restriction site added at its beginning (complete prefix):<br/>GAATTCGCGGCCGCTTCTAGAGAAGGAGGAACTACTATGGCAAAACACCTTTTT<br/>It has a Tm = 68.7&deg;C.<br/><br/>Then I performed two Phusion PCR (one GC and one HF) and one Phusion/RBC PCR to identify the best protocol for SAM synthetase extraction with the new primer.</html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Phusion PCRs|<html><center><table class=\"tn-sp-table\"><tr><th colspan=\"3\">PCR Mixes</th></tr><tr><td style=\"border:none;\"></td><th>Mix HF (1)</th><th>Mix GC (2)</th></tr><tr><td>Phusion Buffer HF</td><td>10&micro;l</td><td>0</td></tr><tr><td>Phusion Buffer GC</td><td>0</td><td>10&micro;l</td></tr><tr><td>dNTPs</td><td colspan=\"2\">1&micro;l</td></tr><tr><td>template</td><td colspan=\"2\">1&micro;l</td></tr><tr><td>Primer Fw</td><td colspan=\"2\" rowspan=\"2\">2.5&micro;l</td></tr><tr><td>Primer Rv</td></tr><tr><td>Phusion pol</td><td colspan=\"2\">0.5&micro;l</td></tr><tr><td>Water</td><td colspan=\"2\">33.5&micro;l</td></tr></table><br/><br/><table class=\"tn-sp-table\"><tr><th colspan=\"4\">Phusion Settings</th></tr><tr><th>Step</th><th>Temperature</th><th>Time</th><th>Go to</th></tr><tr><td>1</td><td>98&deg;C</td><td>30 sec</td><td></td></tr><tr><td>2</td><td>98&deg;C</td><td>10 sec</td><td></td></tr><tr><td>3</td><td>72&deg;C</td><td>35 sec</td><td>step #2, 30 times</td></tr><tr><td>4</td><td>72&deg;C</td><td>10 min</td><td></td></tr><tr><td>5</td><td>4&deg;C</td><td>pause</td><td></td></tr></table></center></html>}}<html></html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Phusion/RBC PCR|<html><center><table class=\"tn-sp-table\"><tr><th colspan=\"2\">PCR Mix</th></tr><tr><td style=\"border:none;\"></td><td>Mix RBC (3)</td></tr><tr><td>Template</td><td>1&micro;l</td></tr><tr><td>dNTPs</td><td>0.5&micro;l</td></tr><tr><td>Primer Fw</td><td rowspan=\"2\">1&micro;l</td></tr><tr><td>Primer Rv</td></tr><tr><td>Buffer RBC</td><td>5&micro;l</td></tr><tr><td>Phusion pol</td><td>0.3&micro;l</td></tr><tr><td>RBC</td><td>0.25&micro;l</td></tr><tr><td>Water</td><td>40.95&micro;l</td></tr></table><br/><br/><table class=\"tn-sp-table\"><tr><th colspan=\"4\">PCR Settings</th></tr><tr><th>Step</th><th>Temperature</th><th>Time</th><th>Go to</th></tr><tr><td>1</td><td>94&deg;C</td><td>2 min</td><td></td></tr><tr><td>2</td><td>94&deg;C</td><td>1 min</td><td></td></tr><tr><td>3</td><td>62.5&deg;C</td><td>1 min</td><td></td></tr><tr><td>4</td><td>72&deg;C</td><td>1 min 9 sec</td><td>step #2, 30 times</td></tr><tr><td>5</td><td>72&deg;C</td><td>7 min</td><td></td></tr><tr><td>6</td><td>4&deg;C</td><td>pause</td><td></td></tr></table></html>}}<html><br/>The products of these three PCRs were then loaded on a 1% agarose gel.</html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Gel|<html><center><table class=\"tn-sp-table\"><tr><th colspan=\"4\">Loading scheme</th></tr><tr><td>1kb ladder</td><td>GC(2)</td><td>HF(1)</td><td>RBC(3)</td></tr></table><img src=\"https://static.igem.org/mediawiki/2013/8/82/Tn-20130710-GG_SAMsynthetase_newPrimers_chosePCR.jpg\" alt=\"Gel\" width=\"450px\"></center></html>}}<html>As showed by the gel, only the Phusion/RBC PCR was successful.<br/><br/><hr><h3>Purifications</h3>Then, I purified the EX-SAMsynthetase-SP sample produced today with the Phusion/RBC PCR, and the 5 R0010 PCR insert that were amplified on </ins><a href=\"https://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/Notebook#tn-post-2013-07-<ins class="diffchange diffchange-inline">02</ins>-gabriele\"><ins class="diffchange diffchange-inline">tuesday 02/07</ins></a><ins class="diffchange diffchange-inline">.<center><table class=\"tn-sp-table\"><tr><th>Sample</th><th>Type</th><th>Quantity</th></tr><tr><td>RBC(3)</td><td>EX-SAMsynthetase-SP</td><td>103.6ng/&micro;l</td></tr><tr><td>HF1</td><td>R0010 insert</td><td>17.5ng/&micro;l</td></tr><tr><td>HF2</td><td>R0010 insert</td><td>19.5ng/&micro;l</td></tr><tr><td>HF3</td><td>R0010 insert</td><td>15.9ng/&micro;l</td></tr><tr><td>G1</td><td>R0010 insert</td><td>17.8ng/&micro;l</td></tr><tr><td>G2</td><td>R0010 insert</td><td>16.3ng/&micro;l</td></tr></table></center><br><hr><h3>OverNight Purification</h3>Then, Viola was so polite to prepare the O/N digestion mixes (<a href=\"https://2013.igem.org/Team:UNITN-Trento/Protocols#Digestion\">with the usual protocol</a>) </ins>and <ins class="diffchange diffchange-inline">incubate </ins>them at <ins class="diffchange diffchange-inline">37</ins>&deg;C. <ins class="diffchange diffchange-inline">An EX-SAMsynthetase-SP sample (RBC#3 from today, 103.6ng/&micro;l) and a linear pSB1C3 sample (G3A from <a href=\"https://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/Notebook#tn-post-2013-07-01-Gabriele\">01/07</a>, 44.8ng/&micro;l) were restricted with XbaI and PstI (Nebuffer2).</html>{{:Team:UNITN-Trento/Templates/Styles/Spoiler|Digestion mixes|<html></ins><center><table class=\"tn-sp-table\"><tr<ins class="diffchange diffchange-inline">><td style=\"border:none;\"></td</ins>><th><ins class="diffchange diffchange-inline">RBC#3</ins></th><th><ins class="diffchange diffchange-inline">G3A</ins></th></tr><tr><td><ins class="diffchange diffchange-inline">Template</ins></td><td><ins class="diffchange diffchange-inline">40&micro;l</td><td>50&micro;l</ins></td></tr><tr><td><ins class="diffchange diffchange-inline">XbaI</ins></td><td <ins class="diffchange diffchange-inline">rowspan=\"2\"</ins>>2<ins class="diffchange diffchange-inline">.5&micro;l</td><td rowspan=\"2\">1.5&micro;l</ins></td></tr><tr><td><ins class="diffchange diffchange-inline">PstI</td></tr><tr><td>NEBuffer </ins>2</td><td><ins class="diffchange diffchange-inline">10&micro;l</td><td>5&micro;l</td></tr><tr><td>BSA</td><td>10&micro;l</td><td>5&micro;l</td></tr><tr><td>Water</td><td>35&micro;l</td><td>0</ins></td></tr></table></center<ins class="diffchange diffchange-inline">></html>}}<html</ins>></html>","tags" : "SAMsynthetase"}</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">12</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" : "gabriele",</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">Plates check</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">Checked out </del>the <del class="diffchange diffchange-inline">plates </del>from <a href=\"https://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/Notebook#tn-post-2013-07-<del class="diffchange diffchange-inline">11</del>-gabriele\"><del class="diffchange diffchange-inline">yesterday</del></a> and <del class="diffchange diffchange-inline">then stocked </del>them at <del class="diffchange diffchange-inline">4</del>&deg;C.<center><table class=\"tn-sp-table\"><tr><th><del class="diffchange diffchange-inline">Plate</del></th><th><del class="diffchange diffchange-inline">#colonies</del></th></tr><tr><td><del class="diffchange diffchange-inline">CTRL</del></td><td><del class="diffchange diffchange-inline">7</del></td></tr><tr><td><del class="diffchange diffchange-inline">1:1</del></td><td>2</td></tr><tr><td><del class="diffchange diffchange-inline">1:</del>2</td><td><del class="diffchange diffchange-inline">1</del></td></tr></table></center></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" : "SAMsynthetase"</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/07/25&diff=273353&oldid=prevGgirelli at 08:29, 3 October 20132013-10-03T08:29:45Z<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">29</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">12</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">fabio</del>",</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> "author" : "<ins class="diffchange diffchange-inline">gabriele</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">the blue ligation: the never ending story !</del>",</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> "title" : "<ins class="diffchange diffchange-inline">Plates check</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"> here we go again!! Today I miniprepped </del>the <del class="diffchange diffchange-inline">third ligation inocula </del>and <del class="diffchange diffchange-inline">the screening as asual wasn’t successful!! I’m kinda bummed!! At list I have my fourth and last ligation</del>: <del class="diffchange diffchange-inline">I made 6 inocula from the plate </del>1:1<del class="diffchange diffchange-inline">. Will see tomorrow!</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">Checked out </ins>the <ins class="diffchange diffchange-inline">plates from <a href=\"https://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/Notebook#tn-post-2013-07-11-gabriele\">yesterday</a> </ins>and <ins class="diffchange diffchange-inline">then stocked them at 4&deg;C.<center><table class=\"tn-sp-table\"><tr><th>Plate</th><th>#colonies</th></tr><tr><td>CTRL</td><td>7</td></tr><tr><td>1</ins>:<ins class="diffchange diffchange-inline">1</td><td>2</td></tr><tr><td></ins>1:<ins class="diffchange diffchange-inline">2</td><td></ins>1<ins class="diffchange diffchange-inline"></td></tr></table></center></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">YF1_FixJ - FixK2</del>"</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> "tags" : "<ins class="diffchange diffchange-inline">SAMsynthetase</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/07/25&diff=110186&oldid=prevGgirelli: moved Team:UNITN-Trento/Notebook/Labposts/124 to Team:UNITN-Trento/Notebook/Labposts/07/252013-09-09T07:26:54Z<p>moved <a href="/Team:UNITN-Trento/Notebook/Labposts/124" class="mw-redirect" title="Team:UNITN-Trento/Notebook/Labposts/124">Team:UNITN-Trento/Notebook/Labposts/124</a> to <a href="/Team:UNITN-Trento/Notebook/Labposts/07/25" title="Team:UNITN-Trento/Notebook/Labposts/07/25">Team:UNITN-Trento/Notebook/Labposts/07/25</a></p>
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<td colspan='1' style="background-color: white; color:black;">Revision as of 07:26, 9 September 2013</td>
</tr></table>Ggirellihttp://2013.igem.org/wiki/index.php?title=Team:UNITN-Trento/Notebook/Labposts/07/25&diff=78619&oldid=prevGgirelli: Created page with "{ "date" : "2013-07-29", "author" : "fabio", "title" : " the blue ligation: the never ending story !", "content" : "<html> here we go again!! Today I miniprepped the third l..."2013-08-19T13:43:20Z<p>Created page with "{ "date" : "2013-07-29", "author" : "fabio", "title" : " the blue ligation: the never ending story !", "content" : "<html> here we go again!! Today I miniprepped the third l..."</p>
<p><b>New page</b></p><div>{<br />
"date" : "2013-07-29",<br />
"author" : "fabio",<br />
"title" : " the blue ligation: the never ending story !",<br />
"content" : "<html> here we go again!! Today I miniprepped the third ligation inocula and the screening as asual wasn’t successful!! I’m kinda bummed!! At list I have my fourth and last ligation: I made 6 inocula from the plate 1:1. Will see tomorrow!</html>",<br />
"tags" : " YF1_FixJ - FixK2"<br />
}</div>Ggirelli