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| − | <html>
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| − | <h1 class="sectionedit1"><a id="interlab_study" name="interlab_study">InterLab Study</a></h1>
| + | |
| − | <div class="level1">
| + | |
| − | <div class="table sectionedit2"><table class="inline">
| + | |
| − | <tr class="row0">
| + | |
| − | <th class="col0"> our construct </th><th class="col1"> BBa </th><th class="col2"> promotor strength </th>
| + | |
| − | </tr>
| + | |
| − | <tr class="row1">
| + | |
| − | <td class="col0"> pILS 1 </td><td class="col1"> J23117 </td><td class="col2"> 162 </td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row2">
| + | |
| − | <td class="col0"> pILS 2 </td><td class="col1"> J23106 </td><td class="col2"> 1185 </td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row3">
| + | |
| − | <td class="col0"> pILS 3 </td><td class="col1"> J23101 </td><td class="col2"> 1791 </td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row4">
| + | |
| − | <td class="col0"> positive </td><td class="col1"> J23151 <br/>
| + | |
| − | 1bp mutant from J23114 </td><td class="col2"> 256 (J23114) </td>
| + | |
| − | </tr>
| + | |
| − | </table></div>
| + | |
| − | <!-- EDIT2 TABLE [31-217] -->
| + | |
| − | </div>
| + | |
| − | <!-- EDIT1 SECTION "InterLab Study" [1-229] -->
| + | |
| − | <h3 class="sectionedit3"><a id="section12082015" name="section12082015">12.08.2015</a></h3>
| + | |
| − | <div class="level3">
| + | |
| − | </div>
| + | |
| − | <h4><a id="top10_pils2_and_pils3_10_c" name="top10_pils2_and_pils3_10_c">TOP10 pILS2 and pILS3 10°C</a></h4>
| + | |
| − | <div class="level4">
| + | |
| − | <ul>
| + | |
| − | <li class="level1"><div class="li"> because not all the colonies grew properly the experiment was repeated for TOP10 pILS2 and pILS3 at 10°C</div>
| + | |
| − | </li>
| + | |
| − | </ul>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT3 SECTION "12.08.2015" [230-397] -->
| + | |
| − | <h3 class="sectionedit4"><a id="section11082015" name="section11082015">11.08.2015</a></h3>
| + | |
| − | <div class="level3">
| + | |
| − | <ul>
| + | |
| − | <li class="level1"><div class="li"> the <a class="urlextern" href="https://2015.igem.org/Tracks/Measurement/InterLab_Protocol" rel="nofollow" target="_Blank" title="https://2015.igem.org/Tracks/Measurement/InterLab_Protocol">protocol on the iGEM website</a> was followed:</div>
| + | |
| − | </li>
| + | |
| − | <li class="level1"><div class="li"> The samples were diluted to OD<sub>600</sub> = 0.5:</div>
| + | |
| − | <ul>
| + | |
| − | <li class="level2"><div class="li"> From all 60 samples the OD<sub>600</sub> was measured using a nanoDrop</div>
| + | |
| − | </li>
| + | |
| − | <li class="level2"><div class="li"> samples were diluted to achieve a final OD<sub>600</sub> of 0.5</div>
| + | |
| − | </li>
| + | |
| − | <li class="level2"><div class="li"> dilution was checked by again measuring the optical density of the samples</div>
| + | |
| − | </li>
| + | |
| − | <li class="level2"><div class="li"> in case of a value outside 0.5 +- 5% the diluation was repeated based on the new values</div>
| + | |
| − | </li>
| + | |
| − | </ul>
| + | |
| − | </li>
| + | |
| − | <li class="level1"><div class="li"> fluorescence was measured using a platereader an an excitation of 485nm with a bandpassfilter of 20nm and emission at 530nm with the filter width.</div>
| + | |
| − | <ul>
| + | |
| − | <li class="level2"><div class="li"> all fluorescence data was measured three times in three 96-well plates (technical replicates)</div>
| + | |
| − | </li>
| + | |
| − | </ul>
| + | |
| − | </li>
| + | |
| − | </ul>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT4 SECTION "11.08.2015" [398-1162] -->
| + | |
| − | <h3 class="sectionedit5"><a id="section10082015" name="section10082015">10.08.2015</a></h3>
| + | |
| − | <div class="level3">
| + | |
| − | <ul>
| + | |
| − | <li class="level1"><div class="li"> AS the BL21 strain carries the same resistance (Cml) as the plasmid does and it wasnot possible to sort out transformed colonies by using the fluorescence as guide we decided not to use BL21 in the study</div>
| + | |
| − | </li>
| + | |
| − | <li class="level1"><div class="li"> from each plate two test tubes with 5 mL LB medium (+Cml) were inoculated</div>
| + | |
| − | </li>
| + | |
| − | <li class="level1"><div class="li"> grown them for 8 h on 37°C, 220 rpm</div>
| + | |
| − | </li>
| + | |
| − | <li class="level1"><div class="li"> after 8 h transfered half of the test tubes to 10°C</div>
| + | |
| − | </li>
| + | |
| − | </ul>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT5 SECTION "10.08.2015" [1163-1570] -->
| + | |
| − | <h3 class="sectionedit6"><a id="section09082015" name="section09082015">09.08.2015</a></h3>
| + | |
| − | <div class="level3">
| + | |
| − | <ul>
| + | |
| − | <li class="level1"><div class="li"> For the other 2 strains: Three colonies of each strain were picked and stricken out on plate</div>
| + | |
| − | </li>
| + | |
| − | <li class="level1"><div class="li"> incubation o/n @ 37°C</div>
| + | |
| − | </li>
| + | |
| − | </ul>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT6 SECTION "09.08.2015" [1571-1717] -->
| + | |
| − | <h3 class="sectionedit7"><a id="section08082015" name="section08082015">08.08.2015</a></h3>
| + | |
| − | <div class="level3">
| + | |
| − | <p>
| + | |
| − | Used <em>E.coli</em> strains are TOP10 (cloning strain) and Bl21pLysS + Arctic Express (expression strain)
| + | |
| − | Each construct was transformed into the different strain.
| + | |
| − | Constructs:
| + | |
| − | </p>
| + | |
| − | <ul>
| + | |
| − | <li class="level1"><div class="li"> Interlab positiv</div>
| + | |
| − | </li>
| + | |
| − | <li class="level1"><div class="li"> Interlab negativ</div>
| + | |
| − | </li>
| + | |
| − | <li class="level1"><div class="li"> pILS 1 (2)</div>
| + | |
| − | </li>
| + | |
| − | <li class="level1"><div class="li"> pILS 2 (2)</div>
| + | |
| − | </li>
| + | |
| − | <li class="level1"><div class="li"> pILS 3 (1)</div>
| + | |
| − | </li>
| + | |
| − | </ul>
| + | |
| − | <p>
| + | |
| − | The antibiotic resistance of the plasmid is chloramphenicol. The expression strain Bl21pLysS has the same resistance. For o/n cultures only the visual positiv colonies (expression of gfp → greenish colonies will be choosen. Due to the fact of having no resistance (TOP10) and another antibiotic resistance (gentamycin) for Arctic Expression the selection by these strain is easier.
| + | |
| − | </p>
| + | |
| − | <ul>
| + | |
| − | <li class="level1"><div class="li"> transformation was performed according to the protocol <a class="wikilink1" href="/igem2015/doku.php?id=protocols:trafo" title="protocols:trafo">Transformation</a></div>
| + | |
| − | </li>
| + | |
| − | </ul>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT7 SECTION "08.08.2015" [1718-2479] -->
| + | |
| − | <h3 class="sectionedit8"><a id="section20150805" name="section20150805">2015/08/05</a></h3>
| + | |
| − | <div class="level3">
| + | |
| − | <p>
| + | |
| − | <strong> Inoculation of 5 ml liquid culture (RE) </strong>
| + | |
| − | </p>
| + | |
| − | <ul>
| + | |
| − | <li class="level1"><div class="li"> pILS_1 (LB_cml)</div>
| + | |
| − | </li>
| + | |
| − | <li class="level1"><div class="li"> pILS_2 (LB_cml)</div>
| + | |
| − | </li>
| + | |
| − | <li class="level1"><div class="li"> pILS_3 (LB_cml)</div>
| + | |
| − | </li>
| + | |
| − | </ul>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT8 SECTION "2015/08/05" [2480-2609] -->
| + | |
| − | <h3 class="sectionedit9"><a id="section20150804" name="section20150804">2015/08/04</a></h3>
| + | |
| − | <div class="level3">
| + | |
| − | <p>
| + | |
| − | <strong> Transformation (RE) </strong>
| + | |
| − | </p>
| + | |
| − | <ul>
| + | |
| − | <li class="level1"><div class="li"> pILS_1</div>
| + | |
| − | </li>
| + | |
| − | <li class="level1"><div class="li"> pILS_2</div>
| + | |
| − | </li>
| + | |
| − | <li class="level1"><div class="li"> pILS_3</div>
| + | |
| − | </li>
| + | |
| − | </ul>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT9 SECTION "2015/08/04" [2610-2693] -->
| + | |
| − | <h3 class="sectionedit10"><a id="section20150803" name="section20150803">2015/08/03</a></h3>
| + | |
| − | <div class="level3">
| + | |
| − | <p>
| + | |
| − | <strong>Test digest: Interlab Study (LS)</strong>
| + | |
| − | </p>
| + | |
| − | <ul>
| + | |
| − | <li class="level1"><div class="li"> Interlab Study: pILS1, 2 and 3–>EcoRI HF and SpeI HF</div>
| + | |
| − | </li>
| + | |
| − | </ul>
| + | |
| − | <div class="table sectionedit11"><table class="inline">
| + | |
| − | <tr class="row0">
| + | |
| − | <th class="col0" colspan="2">6xMasterMix</th>
| + | |
| − | </tr>
| + | |
| − | <tr class="row1">
| + | |
| − | <td class="col0">6µl</td><td class="col1">EcoRI HF</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row2">
| + | |
| − | <td class="col0">6µl</td><td class="col1">SpeI Hf</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row3">
| + | |
| − | <td class="col0">12µl</td><td class="col1">Cut smart</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row4">
| + | |
| − | <td class="col0">66µl</td><td class="col1">dH2O</td>
| + | |
| − | </tr>
| + | |
| − | </table></div>
| + | |
| − | <!-- EDIT11 TABLE [2813-2889] --><div class="thumb2 trien" style="width:410px"><div class="thumbinner"><a class="media" href="https://static.igem.org/mediawiki/2015/7/78/Freiburg_labjournal-cloning-interlabstudy.png" title="labjournal:cloning:interlabstudy.png"><img alt="" class="mediabox2" src="https://static.igem.org/mediawiki/2015/7/78/Freiburg_labjournal-cloning-interlabstudy.png" width="400"/></a><div class="thumbcaption"><div class="magnify"><a class="internal" href="https://static.igem.org/mediawiki/2015/7/78/Freiburg_labjournal-cloning-interlabstudy.png" title="vergrößern"><img alt="" height="11" src="/igem2015/lib/plugins/imagebox/magnify-clip.png" width="15"/></a></div></div></div></div>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT10 SECTION "2015/08/03" [2694-2940] -->
| + | |
| − | <h3 class="sectionedit12"><a id="section20150802" name="section20150802">2015/08/02</a></h3>
| + | |
| − | <div class="level3">
| + | |
| − | <p>
| + | |
| − | <strong>Picked colonies from ligation plate (interlab study) (?)</strong>
| + | |
| − | </p>
| + | |
| − | <p>
| + | |
| − | <strong>Miniprep: pILS1,2,3 (JN)</strong>
| + | |
| − | </p>
| + | |
| − | <ul>
| + | |
| − | <li class="level1"><div class="li"> qiagen kit</div>
| + | |
| − | </li>
| + | |
| − | <li class="level1"><div class="li"> eluted in 30µl dH2O</div>
| + | |
| − | </li>
| + | |
| − | </ul>
| + | |
| − | <div class="table sectionedit13"><table class="inline">
| + | |
| − | <tr class="row0">
| + | |
| − | <th class="col0">plasmid</th><th class="col1">concentration</th>
| + | |
| − | </tr>
| + | |
| − | <tr class="row1">
| + | |
| − | <td class="col0">pILS1 (1)</td><td class="col1">84,4</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row2">
| + | |
| − | <td class="col0">pILS1 (2)</td><td class="col1">51,7</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row3">
| + | |
| − | <td class="col0">pILS2 (1)</td><td class="col1">60,7</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row4">
| + | |
| − | <td class="col0">pILS2 (2)</td><td class="col1">107,3</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row5">
| + | |
| − | <td class="col0">pILS3 (1)</td><td class="col1">11,2</td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row6">
| + | |
| − | <td class="col0">pILS3 (2)</td><td class="col1">5,1</td>
| + | |
| − | </tr>
| + | |
| − | </table></div>
| + | |
| − | <!-- EDIT13 TABLE [3096-3221] -->
| + | |
| − | </div>
| + | |
| − | <!-- EDIT12 SECTION "2015/08/02" [2941-3223] -->
| + | |
| − | <h3 class="sectionedit14"><a id="section20150801" name="section20150801">2015/08/01</a></h3>
| + | |
| − | <div class="level3">
| + | |
| − | <p>
| + | |
| − | <strong> Digest of the ILS plasmids (RE) </strong>
| + | |
| − | </p>
| + | |
| − | <ul>
| + | |
| − | <li class="level1"><div class="li"> Bba_K823013 with SpeI and PstI-HF</div>
| + | |
| − | </li>
| + | |
| − | <li class="level1"><div class="li"> Bba_K823008 with SpeI and PstI-HF</div>
| + | |
| − | </li>
| + | |
| − | <li class="level1"><div class="li"> Bba_K823005 with SpeI and PstI-HF</div>
| + | |
| − | </li>
| + | |
| − | <li class="level1"><div class="li"> Bba_I13504 with XbaI and PstI-HF</div>
| + | |
| − | </li>
| + | |
| − | </ul>
| + | |
| − | <ul>
| + | |
| − | <li class="level1"><div class="li"> 1 µl of each restr. enzyme</div>
| + | |
| − | </li>
| + | |
| − | <li class="level1"><div class="li"> 5 µl CutSmart </div>
| + | |
| − | </li>
| + | |
| − | <li class="level1"><div class="li"> 10 µl DNA</div>
| + | |
| − | </li>
| + | |
| − | <li class="level1"><div class="li"> 33 µl dH2O</div>
| + | |
| − | </li>
| + | |
| − | <li class="level1"><div class="li"> incubated for 1 h at 37°C</div>
| + | |
| − | </li>
| + | |
| − | </ul>
| + | |
| − | <p>
| + | |
| − | analysis on 1 % agarose gel:
| + | |
| − | </p>
| + | |
| − | <div class="thumb2 trien" style="width:410px"><div class="thumbinner"><a class="media" href="https://static.igem.org/mediawiki/2015/a/ac/Freiburg_labjournal-cloning-2015_08_01digestpils_spe_xba_pst.png" title="labjournal:cloning:2015_08_01digestpils_spe_xba_pst.png"><img alt="" class="mediabox2" src="https://static.igem.org/mediawiki/2015/a/ac/Freiburg_labjournal-cloning-2015_08_01digestpils_spe_xba_pst.png" width="400"/></a><div class="thumbcaption"><div class="magnify"><a class="internal" href="https://static.igem.org/mediawiki/2015/a/ac/Freiburg_labjournal-cloning-2015_08_01digestpils_spe_xba_pst.png" title="vergrößern"><img alt="" height="11" src="/igem2015/lib/plugins/imagebox/magnify-clip.png" width="15"/></a></div></div></div></div><hr/>
| + | |
| − | <p>
| + | |
| − | <strong>Ligation (RE)</strong>
| + | |
| − | </p>
| + | |
| − | <ul>
| + | |
| − | <li class="level1"><div class="li"> InterLab study:</div>
| + | |
| − | </li>
| + | |
| − | </ul>
| + | |
| − | <div class="table sectionedit15"><table class="inline">
| + | |
| − | <tr class="row0">
| + | |
| − | <td class="col0"> Backbone </td><td class="col1"> Bba_K823013 </td><td class="col2"> Bba_K823008 </td><td class="col3"> Bba_K82_3005 </td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row1">
| + | |
| − | <td class="col0"> Backbone amount [µl] </td><td class="col1"> 2.17 </td><td class="col2"> 1 </td><td class="col3"> 2.08 </td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row2">
| + | |
| − | <td class="col0"> GFP (Bba_I13504) amount [µl] </td><td class="col1"> 3.09 </td><td class="col2"> 3.09 </td><td class="col3"> 3.09 </td>
| + | |
| − | </tr>
| + | |
| − | </table></div>
| + | |
| − | <!-- EDIT15 TABLE [3698-3852] -->
| + | |
| − | <p>
| + | |
| − | <strong> Transformation of the ligation products (5 µl) into E.coli TOP10 (RE) </strong>
| + | |
| − | </p>
| + | |
| − | <ul>
| + | |
| − | <li class="level1"><div class="li"> pILS1-3 were plated on LB-cml</div>
| + | |
| − | </li>
| + | |
| − | </ul>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT14 SECTION "2015/08/01" [3224-3966] -->
| + | |
| − | <h3 class="sectionedit16"><a id="section26072015" name="section26072015">26.07.2015</a></h3>
| + | |
| − | <div class="level3">
| + | |
| − | <ul>
| + | |
| − | <li class="level1"><div class="li"> from the first miniprep another try for GFP (miniprep with 105.1 ng/µL) digest was done</div>
| + | |
| − | </li>
| + | |
| − | </ul>
| + | |
| − | <div class="table sectionedit17"><table class="inline">
| + | |
| − | <tr class="row0">
| + | |
| − | <th class="col0"> </th><th class="col1"> volume [µL] </th>
| + | |
| − | </tr>
| + | |
| − | <tr class="row1">
| + | |
| − | <td class="col0"> DNA </td><td class="col1"> 10 </td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row2">
| + | |
| − | <td class="col0"> 10x cutSmart </td><td class="col1"> 5 µL </td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row3">
| + | |
| − | <td class="col0"> Xba1 </td><td class="col1"> 1 µL </td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row4">
| + | |
| − | <td class="col0"> Pst1-HF </td><td class="col1"> 1µL </td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row5">
| + | |
| − | <td class="col0"> water </td><td class="col1"> 33 µL </td>
| + | |
| − | </tr>
| + | |
| − | </table></div>
| + | |
| − | <!-- EDIT17 TABLE [4082-4193] --><ul>
| + | |
| − | <li class="level1"><div class="li"> <a class="urlextern" href="http://nebcloner.neb.com/#!/protocol/re/double/XbaI,PstI-HF" rel="nofollow" target="_Blank" title="http://nebcloner.neb.com/#!/protocol/re/double/XbaI,PstI-HF">NEB Cloner</a> proposed 5-15 min @ 37°C but digest over night should also be possible</div>
| + | |
| − | </li>
| + | |
| − | <li class="level1"><div class="li"> digest was carried out for 2.5h @ 37 in Thermocycler</div>
| + | |
| − | </li>
| + | |
| − | <li class="level1"><div class="li"> sample was loaded on a free lane of a before used 1% agarose gel</div>
| + | |
| − | <ul>
| + | |
| − | <li class="level2"><div class="li"> 5µL 1kb ladder was used</div>
| + | |
| − | </li>
| + | |
| − | </ul>
| + | |
| − | </li>
| + | |
| − | </ul>
| + | |
| − | </div>
| + | |
| − | <h4><a id="results" name="results">Results</a></h4>
| + | |
| − | <div class="level4">
| + | |
| − | <div class="thumb2 trien" style="width:35px"><div class="thumbinner"><a class="media" href="https://static.igem.org/mediawiki/2015/5/5c/Freiburg_labjournal-interlab-20150726_interlab_gfp_digest.jpg" title="labjournal:interlab:20150726_interlab_gfp_digest.jpg"><img alt="" class="mediabox2" src="https://static.igem.org/mediawiki/2015/5/5c/Freiburg_labjournal-interlab-20150726_interlab_gfp_digest.jpg" width="25"/></a><div class="thumbcaption"><div class="magnify"><a class="internal" href="https://static.igem.org/mediawiki/2015/5/5c/Freiburg_labjournal-interlab-20150726_interlab_gfp_digest.jpg" title="vergrößern"><img alt="" height="11" src="/igem2015/lib/plugins/imagebox/magnify-clip.png" width="15"/></a></div>Lane of digested GFP.</div></div></div><ul>
| + | |
| − | <li class="level1"><div class="li"> Only one band is visible so the digest was not succesfull</div>
| + | |
| − | </li>
| + | |
| − | <li class="level1"><div class="li"> Additionally the 1kb-ladder was not visible on the gel.</div>
| + | |
| − | </li>
| + | |
| − | </ul>
| + | |
| − | <p>
| + | |
| − | <p><div class="noteimportant">Before further experiments are carried out, the DNA from the second miniprep has to be sequenced to prevent the right digest of the wrong DNA!
| + | |
| − | </div></p>
| + | |
| − | </p>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT16 SECTION "26.07.2015" [3967-4896] -->
| + | |
| − | <h3 class="sectionedit18"><a id="section25072015" name="section25072015">25.07.2015</a></h3>
| + | |
| − | <div class="level3">
| + | |
| − | <ul>
| + | |
| − | <li class="level1"><div class="li"> A miniprep for all 4 InterLabStudy-constructs was carried out</div>
| + | |
| − | </li>
| + | |
| − | </ul>
| + | |
| − | <div class="table sectionedit19"><table class="inline">
| + | |
| − | <tr class="row0">
| + | |
| − | <th class="col0"> construct </th><th class="col1"> yield [ng/µL] </th>
| + | |
| − | </tr>
| + | |
| − | <tr class="row1">
| + | |
| − | <td class="col0"> BBa_I13504 </td><td class="col1"> 111.9 </td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row2">
| + | |
| − | <td class="col0"> BBa_K823005 </td><td class="col1"> 176.6 </td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row3">
| + | |
| − | <td class="col0"> BBa_K823008 </td><td class="col1"> 76.3 </td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row4">
| + | |
| − | <td class="col0"> BBa_K8230 </td><td class="col1"> 109.0 </td>
| + | |
| − | </tr>
| + | |
| − | </table></div>
| + | |
| − | <!-- EDIT19 TABLE [4986-5108] --><ul>
| + | |
| − | <li class="level1"><div class="li"> From each o/n culture a plate culture was stroken out to have cells for further minipreps</div>
| + | |
| − | </li>
| + | |
| − | </ul>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT18 SECTION "25.07.2015" [4897-5204] -->
| + | |
| − | <h3 class="sectionedit20"><a id="section24072015" name="section24072015">24.07.2015</a></h3>
| + | |
| − | <div class="level3">
| + | |
| − | <ul>
| + | |
| − | <li class="level1"><div class="li"> from all four constructs colonies still stored @ 4°C cells were taken and put into 3 mL LB-medium + Cml</div>
| + | |
| − | </li>
| + | |
| − | <li class="level1"><div class="li"> cells were grown o/n for miniprep the next day</div>
| + | |
| − | </li>
| + | |
| − | </ul>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT20 SECTION "24.07.2015" [5205-5385] -->
| + | |
| − | <h3 class="sectionedit21"><a id="section23072015" name="section23072015">23.07.2015</a></h3>
| + | |
| − | <div class="level3">
| + | |
| − | <ul>
| + | |
| − | <li class="level1"><div class="li"> samples were digested with the right volumes</div>
| + | |
| − | </li>
| + | |
| − | </ul>
| + | |
| − | <div class="table sectionedit22"><table class="inline">
| + | |
| − | <tr class="row0">
| + | |
| − | <th class="col0"> </th><th class="col1"> K823005 </th><th class="col2"> K823008 </th><th class="col3"> K823013-2 </th><th class="col4"> I13504-1 </th>
| + | |
| − | </tr>
| + | |
| − | <tr class="row1">
| + | |
| − | <td class="col0"> Plasmid </td><td class="col1"> 42 µL </td><td class="col2"> 30 µL </td><td class="col3"> 28 µL </td><td class="col4"> 30 µL </td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row2">
| + | |
| − | <td class="col0"> Pst1 </td><td class="col1"> 1 µL </td><td class="col2"> 1 µL </td><td class="col3"> 1 µL </td><td class="col4"> 1 µL </td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row3">
| + | |
| − | <td class="col0"> Spe1 </td><td class="col1"> 1 µL </td><td class="col2">1 µL </td><td class="col3">1 µL </td><td class="col4"> </td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row4">
| + | |
| − | <td class="col0">Xba1 </td><td class="col1"> </td><td class="col2"> </td><td class="col3"> </td><td class="col4"> 1 µL </td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row5">
| + | |
| − | <td class="col0"> 5x buffer </td><td class="col1"> 5 µL </td><td class="col2"> 5 µL </td><td class="col3"> 5 µL </td><td class="col4"> 5 µL </td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row6">
| + | |
| − | <td class="col0"> <em>aqua dest</em> </td><td class="col1"> 1 µL </td><td class="col2"> 13 µL </td><td class="col3"> 15 µL </td><td class="col4"> 13 µL </td>
| + | |
| − | </tr>
| + | |
| − | </table></div>
| + | |
| − | <!-- EDIT22 TABLE [5457-5746] --><ul>
| + | |
| − | <li class="level1"><div class="li"> 1% agarose gel was loaded and run 45 min @ 110V</div>
| + | |
| − | </li>
| + | |
| − | </ul>
| + | |
| − | <div class="thumb2 trien" style="width:410px"><div class="thumbinner"><a class="media" href="https://static.igem.org/mediawiki/2015/9/9e/Freiburg_labjournal-interlab-20150723_interlab_digest1.png" title="labjournal:interlab:20150723_interlab_digest1.png"><img alt="" class="mediabox2" src="https://static.igem.org/mediawiki/2015/9/9e/Freiburg_labjournal-interlab-20150723_interlab_digest1.png" width="400"/></a><div class="thumbcaption"><div class="magnify"><a class="internal" href="https://static.igem.org/mediawiki/2015/9/9e/Freiburg_labjournal-interlab-20150723_interlab_digest1.png" title="vergrößern"><img alt="" height="11" src="/igem2015/lib/plugins/imagebox/magnify-clip.png" width="15"/></a></div>Digest of the samples with Pst1 and Spe1 (for the backbones) and Xba1 (for the insert) respectively. Sample loading should have been all three backbones and then the insert, but samples seem to be swapped.</div></div></div>
| + | |
| − | </div>
| + | |
| − | <h4><a id="results1" name="results1">Results</a></h4>
| + | |
| − | <div class="level4">
| + | |
| − | <ul>
| + | |
| − | <li class="level1"><div class="li"> samples seem to be swapped</div>
| + | |
| − | <ul>
| + | |
| − | <li class="level2"><div class="li"> The GFP insert was assigned to the first construct with about kbp length → There the digest may not have worked and therfore only one band is visible</div>
| + | |
| − | </li>
| + | |
| − | <li class="level2"><div class="li"> The other bands were cut out and marked as Insert 1 to 3 because assignment to the different samples was not clear.</div>
| + | |
| − | </li>
| + | |
| − | <li class="level2"><div class="li"> It is additionally unclear, if there has been succesfull digestion because the loss of 35 bp can't be seen on the gel</div>
| + | |
| − | </li>
| + | |
| − | </ul>
| + | |
| − | </li>
| + | |
| − | <li class="level1"><div class="li"> The GFP-insert digest will be done again</div>
| + | |
| − | </li>
| + | |
| − | <li class="level1"><div class="li"> Backbone samples were stored @ 4°C</div>
| + | |
| − | </li>
| + | |
| − | </ul>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT21 SECTION "23.07.2015" [5386-6604] -->
| + | |
| − | <h3 class="sectionedit23"><a id="section22072015" name="section22072015">22.07.2015</a></h3>
| + | |
| − | <div class="level3">
| + | |
| − | <ul>
| + | |
| − | <li class="level1"><div class="li"> samples were loaded on a 1% <a class="wikilink1" href="/igem2015/doku.php?id=protocols:agarose-gel" title="protocols:agarose-gel">agarose gel</a> and run @ 110V for 30 min</div>
| + | |
| − | </li>
| + | |
| − | <li class="level1"><div class="li"> there were no bands visible</div>
| + | |
| − | </li>
| + | |
| − | </ul>
| + | |
| − | <div class="thumb2 trien" style="width:410px"><div class="thumbinner"><a class="media" href="https://static.igem.org/mediawiki/2015/4/4e/Freiburg_labjournal-interlab-20150722_interlab_digest.jpg" title="labjournal:interlab:20150722_interlab_digest.jpg"><img alt="" class="mediabox2" src="https://static.igem.org/mediawiki/2015/4/4e/Freiburg_labjournal-interlab-20150722_interlab_digest.jpg" width="400"/></a><div class="thumbcaption"><div class="magnify"><a class="internal" href="https://static.igem.org/mediawiki/2015/4/4e/Freiburg_labjournal-interlab-20150722_interlab_digest.jpg" title="vergrößern"><img alt="" height="11" src="/igem2015/lib/plugins/imagebox/magnify-clip.png" width="15"/></a></div>digest of the InterLab constructs failed.</div></div></div>
| + | |
| − | <p>
| + | |
| − | <p><div class="noteimportant">Next time keep track of the differences between ng and µg!
| + | |
| − | </div></p>
| + | |
| − | </p>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT23 SECTION "22.07.2015" [6605-6942] -->
| + | |
| − | <h3 class="sectionedit24"><a id="section21072015" name="section21072015">21.07.2015</a></h3>
| + | |
| − | <div class="level3">
| + | |
| − | <ul>
| + | |
| − | <li class="level1"><div class="li"> constructs were diluted to reach final concentration of 1µg/µL</div>
| + | |
| − | </li>
| + | |
| − | </ul>
| + | |
| − | <div class="table sectionedit25"><table class="inline">
| + | |
| − | <tr class="row0">
| + | |
| − | <th class="col0"> starting construct </th><th class="col1"> dilution </th>
| + | |
| − | </tr>
| + | |
| − | <tr class="row1">
| + | |
| − | <td class="col0"> Bba_K823005 46.4 µg/µL </td><td class="col1"> 45.4 µL <em>aqua dest</em> + 1µL </td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row2">
| + | |
| − | <td class="col0"> Bba_K823008 67.9 µg/µL </td><td class="col1"> 66.9 µL <em>aqua dest</em> + 1µL </td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row3">
| + | |
| − | <td class="col0"> Bba_K823013-2 72.7 µg/µL </td><td class="col1"> 71.7 µL <em>aqua dest</em> + 1µL </td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row4">
| + | |
| − | <td class="col0"> Bba_I13504-1 66.8 µg/µL </td><td class="col1"> 65.8 µL <em>aqua dest</em> + 1µL </td>
| + | |
| − | </tr>
| + | |
| − | </table></div>
| + | |
| − | <!-- EDIT25 TABLE [7035-7315] --><ul>
| + | |
| − | <li class="level1"><div class="li"> digestion was carried out for 1.5 h @ 37°C following the scheme and the <a class="wikilink1" href="/igem2015/doku.php?id=protocols:restriction" title="protocols:restriction">protocol</a></div>
| + | |
| − | </li>
| + | |
| − | </ul>
| + | |
| − | <div class="table sectionedit26"><table class="inline">
| + | |
| − | <tr class="row0">
| + | |
| − | <th class="col0 leftalign"> </th><th class="col1"> K823005 </th><th class="col2"> K823008 </th><th class="col3"> K823013 </th><th class="col4"> I13504 </th>
| + | |
| − | </tr>
| + | |
| − | <tr class="row1">
| + | |
| − | <td class="col0"> Plasmid </td><td class="col1"> 3 µL </td><td class="col2"> 3 µL </td><td class="col3"> 3 µL </td><td class="col4"> 3 µL </td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row2">
| + | |
| − | <td class="col0"> PstI </td><td class="col1"> 1µL </td><td class="col2"> 1µL </td><td class="col3"> 1µL </td><td class="col4"> 1µL </td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row3">
| + | |
| − | <td class="col0"> SpeI </td><td class="col1"> 1µL </td><td class="col2"> 1µL </td><td class="col3"> 1µL </td><td class="col4"> </td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row4">
| + | |
| − | <td class="col0"> XbaI </td><td class="col1"> </td><td class="col2"> </td><td class="col3"> </td><td class="col4"> 1µL </td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row5">
| + | |
| − | <td class="col0"> 5x buffer </td><td class="col1"> 5µL </td><td class="col2"> 5µL </td><td class="col3"> 5µL </td><td class="col4"> 5µL </td>
| + | |
| − | </tr>
| + | |
| − | <tr class="row6">
| + | |
| − | <td class="col0"> <em>aqua dest</em> </td><td class="col1"> 40µL </td><td class="col2"> 40µL </td><td class="col3"> 40µL </td><td class="col4"> 40µL </td>
| + | |
| − | </tr>
| + | |
| − | </table></div>
| + | |
| − | <!-- EDIT26 TABLE [7430-7701] --><ul>
| + | |
| − | <li class="level1"><div class="li"> after digestion enzymes were inactivated by 2min 80°C treatment</div>
| + | |
| − | </li>
| + | |
| − | <li class="level1"><div class="li"> samples were stored @ 4°C</div>
| + | |
| − | </li>
| + | |
| − | </ul>
| + | |
| − | </div>
| + | |
| − | <!-- EDIT24 SECTION "21.07.2015" [6943-] -->
| + | |
| − | </html>
| + | |