Difference between revisions of "Team:Freiburg/Labjournals/Cloning/September"

 
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<h3 class="sectionedit1">2015/09/01</h3>
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      <a style="color:#FFF" href="https://2015.igem.org/Team:Freiburg/Labjournals"> << Back</a>
 
</div>
 
</div>
<!-- EDIT1 SECTION "2015/09/01" [1-22] -->
+
<!--========= END: GoBack button ==========-->
<h3 class="sectionedit2">2015/09/02</h3>
+
 
 +
 
 +
<div class="content_box">
 +
 
 +
<h3 class="sectionedit1">2015/09/02</h3>
 
<div class="level3">
 
<div class="level3">
 
<p>
 
<p>
 
<strong>Digest: pOP, pILS3 and K592009 with EcoRI (LS)</strong>
 
<strong>Digest: pOP, pILS3 and K592009 with EcoRI (LS)</strong>
 
</p>
 
</p>
<div class="table sectionedit3"><table class="inline">
+
<div class="table sectionedit2"><table class="inline">
 
<tr class="row0">
 
<tr class="row0">
 
<th class="col0">amount</th><th class="col1">ingredient</th>
 
<th class="col0">amount</th><th class="col1">ingredient</th>
Line 38: Line 60:
 
</tr>
 
</tr>
 
</table></div>
 
</table></div>
<!-- EDIT3 TABLE [96-180] --><ul>
+
<!-- EDIT2 TABLE [75-159] --><ul>
 
<li class="level1"><div class="li"> incubation at 37°C, ~1h</div>
 
<li class="level1"><div class="li"> incubation at 37°C, ~1h</div>
 
</li>
 
</li>
Line 51: Line 73:
 
</li>
 
</li>
 
</ul>
 
</ul>
<div class="table sectionedit4"><table class="inline">
+
<div class="table sectionedit3"><table class="inline">
 
<tr class="row0">
 
<tr class="row0">
 
<th class="col0"> </th><th class="col1"> concentration [ng/µl]</th>
 
<th class="col0"> </th><th class="col1"> concentration [ng/µl]</th>
Line 65: Line 87:
 
</tr>
 
</tr>
 
</table></div>
 
</table></div>
<!-- EDIT4 TABLE [298-368] -->
+
<!-- EDIT3 TABLE [277-347] -->
 
<p>
 
<p>
 
<strong> Digest of pOP, pILS3 and BBa_K592009 with PstI (JN) </strong>
 
<strong> Digest of pOP, pILS3 and BBa_K592009 with PstI (JN) </strong>
 
</p>
 
</p>
<div class="table sectionedit5"><table class="inline">
+
<div class="table sectionedit4"><table class="inline">
 
<tr class="row0">
 
<tr class="row0">
 
<th class="col0"> ingredient</th><th class="col1"> amount [µl]</th>
 
<th class="col0"> ingredient</th><th class="col1"> amount [µl]</th>
Line 86: Line 108:
 
</tr>
 
</tr>
 
</table></div>
 
</table></div>
<!-- EDIT5 TABLE [428-510] --><ul>
+
<!-- EDIT4 TABLE [407-489] --><ul>
 
<li class="level1"><div class="li"> 37°C for 1h</div>
 
<li class="level1"><div class="li"> 37°C for 1h</div>
 
</li>
 
</li>
Line 92: Line 114:
 
</li>
 
</li>
 
</ul>
 
</ul>
 +
<div class="thumb2 trien" style="width:410px"><div class="thumbinner"><a class="media" href="https://static.igem.org/mediawiki/2015/2/2e/Freiburg_labjournal-cloning-2015_09_02_digestpop_i13504_k592009.png" title="labjournal:cloning:2015_09_02_digestpop_i13504_k592009.png"><img alt="" class="mediabox2" src="https://static.igem.org/mediawiki/2015/2/2e/Freiburg_labjournal-cloning-2015_09_02_digestpop_i13504_k592009.png" width="400"/></a><div class="thumbcaption"><div class="magnify"><a class="internal" href="https://static.igem.org/mediawiki/2015/2/2e/Freiburg_labjournal-cloning-2015_09_02_digestpop_i13504_k592009.png" title="vergrößern"><img alt="" height="11" src="/igem2015/lib/plugins/imagebox/magnify-clip.png" width="15"/></a></div>Expected fragment sizes: <strong>pOP</strong> ~5000bp, <strong>BBa_I13504</strong> ~700bp, <strong>BBa_K592009</strong> ~700bp</div></div></div><hr/>
 
<p>
 
<p>
 
<strong> Gel-ex of BBa_I13504 (JN) </strong>
 
<strong> Gel-ex of BBa_I13504 (JN) </strong>
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<strong> Repetition of pOP Digest with EcoRI (JN) </strong>
 
<strong> Repetition of pOP Digest with EcoRI (JN) </strong>
 
</p>
 
</p>
<div class="table sectionedit6"><table class="inline">
+
<div class="table sectionedit5"><table class="inline">
 
<tr class="row0">
 
<tr class="row0">
 
<th class="col0"> ingredient</th><th class="col1"> amount [µl]</th>
 
<th class="col0"> ingredient</th><th class="col1"> amount [µl]</th>
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</tr>
 
</tr>
 
</table></div>
 
</table></div>
<!-- EDIT6 TABLE [712-780] --><ul>
+
<!-- EDIT5 TABLE [855-923] --><ul>
 
<li class="level1"><div class="li"> 37°C for 1h</div>
 
<li class="level1"><div class="li"> 37°C for 1h</div>
 
</li>
 
</li>
Line 141: Line 164:
 
<strong> pOP digest with PstI </strong>
 
<strong> pOP digest with PstI </strong>
 
</p>
 
</p>
<div class="table sectionedit7"><table class="inline">
+
<div class="table sectionedit6"><table class="inline">
 
<tr class="row0">
 
<tr class="row0">
 
<th class="col0"> ingredient</th><th class="col1"> amount [µl]</th>
 
<th class="col0"> ingredient</th><th class="col1"> amount [µl]</th>
Line 158: Line 181:
 
</tr>
 
</tr>
 
</table></div>
 
</table></div>
<!-- EDIT7 TABLE [934-1004] --><ul>
+
<!-- EDIT6 TABLE [1077-1147] --><ul>
 
<li class="level1"><div class="li"> 37°C for 1h</div>
 
<li class="level1"><div class="li"> 37°C for 1h</div>
 
</li>
 
</li>
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</li>
 
</li>
 
</ul>
 
</ul>
 +
<div class="thumb2 trien" style="width:310px"><div class="thumbinner"><a class="media" href="https://static.igem.org/mediawiki/2015/9/97/Freiburg_labjournal-cloning-2015_09_02_digestpop.png" title="labjournal:cloning:2015_09_02_digestpop.png"><img alt="" class="mediabox2" src="https://static.igem.org/mediawiki/2015/9/97/Freiburg_labjournal-cloning-2015_09_02_digestpop.png" width="300"/></a><div class="thumbcaption"><div class="magnify"><a class="internal" href="https://static.igem.org/mediawiki/2015/9/97/Freiburg_labjournal-cloning-2015_09_02_digestpop.png" title="vergrößern"><img alt="" height="11" src="/igem2015/lib/plugins/imagebox/magnify-clip.png" width="15"/></a></div>Expected fragment size: ~5000bp</div></div></div><hr/>
 
<p>
 
<p>
 
<strong> Gel-ex of pOP (JN) </strong>
 
<strong> Gel-ex of pOP (JN) </strong>
 
</p>
 
</p>
 +
<ul>
 +
<li class="level1"><div class="li"> Qiagen kit</div>
 +
</li>
 +
<li class="level1"><div class="li"> eluted in 20µl dH2O</div>
 +
</li>
 +
<li class="level1"><div class="li"> concentration: 6.4ng/µl</div>
 +
</li>
 +
</ul>
 
<p>
 
<p>
 
<strong> Ligation of pOP+BBa_I13504 (JN) </strong>
 
<strong> Ligation of pOP+BBa_I13504 (JN) </strong>
 
</p>
 
</p>
 +
<ul>
 +
<li class="level1"><div class="li"> Backbone: 3.4 µl</div>
 +
</li>
 +
<li class="level1"><div class="li"> BBa_I13504: 10.5 µl</div>
 +
</li>
 +
<li class="level1"><div class="li"> buffer: 2 µl</div>
 +
</li>
 +
<li class="level1"><div class="li"> T4 ligase: 1 µl</div>
 +
</li>
 +
<li class="level1"><div class="li"> dH2O: 3.1 µl</div>
 +
</li>
 +
</ul>
 
<p>
 
<p>
 
<strong> Trafo: Ligation of pOP BBA_I13504 in <em>E.coli</em> T10 (LS) </strong>
 
<strong> Trafo: Ligation of pOP BBA_I13504 in <em>E.coli</em> T10 (LS) </strong>
 
</p>
 
</p>
 
<ul>
 
<ul>
<li class="level1"><div class="li"> 10µl of ligation</div>
+
<li class="level1"><div class="li"> 7 µl of ligation</div>
 
</li>
 
</li>
 
<li class="level1"><div class="li"> transformation according to protocol</div>
 
<li class="level1"><div class="li"> transformation according to protocol</div>
Line 180: Line 224:
 
</ul>
 
</ul>
 
</div>
 
</div>
<!-- EDIT2 SECTION "2015/09/02" [23-1246] -->
+
<!-- EDIT1 SECTION "2015/09/02" [2-1655] -->
<h3 class="sectionedit8">2015/09/03</h3>
+
<h3 class="sectionedit7">2015/09/03</h3>
 
<div class="level3">
 
<div class="level3">
 
<p>
 
<p>
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</ul>
 
</ul>
 
</div>
 
</div>
<!-- EDIT8 SECTION "2015/09/03" [1247-1511] -->
+
<!-- EDIT7 SECTION "2015/09/03" [1656-1920] -->
<h3 class="sectionedit9">2015/09/04</h3>
+
<h3 class="sectionedit8">2015/09/04</h3>
 
<div class="level3">
 
<div class="level3">
 
<p>
 
<p>
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</li>
 
</li>
 
</ul>
 
</ul>
<div class="table sectionedit10"><table class="inline">
+
<div class="table sectionedit9"><table class="inline">
 
<tr class="row0">
 
<tr class="row0">
 
<th class="col0"> </th><th class="col1"> concentration [ng/µl]</th>
 
<th class="col0"> </th><th class="col1"> concentration [ng/µl]</th>
Line 232: Line 276:
 
</tr>
 
</tr>
 
</table></div>
 
</table></div>
<!-- EDIT10 TABLE [1741-1850] --><ul>
+
<!-- EDIT9 TABLE [2150-2259] --><ul>
 
<li class="level1"><div class="li"> sample 9 and 11 were sent in for sequencing</div>
 
<li class="level1"><div class="li"> sample 9 and 11 were sent in for sequencing</div>
 
</li>
 
</li>
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</ul>
 
</ul>
 
</div>
 
</div>
<!-- EDIT9 SECTION "2015/09/04" [1512-2067] -->
+
<!-- EDIT8 SECTION "2015/09/04" [1921-2476] -->
<h3 class="sectionedit11">2015/09/05</h3>
+
<h3 class="sectionedit10">2015/09/05</h3>
 
<div class="level3">
 
<div class="level3">
 
<p>
 
<p>
Line 265: Line 309:
 
<strong>Digest: pOP and K592009 with EcoRI (LS)</strong>
 
<strong>Digest: pOP and K592009 with EcoRI (LS)</strong>
 
</p>
 
</p>
<div class="table sectionedit12"><table class="inline">
+
<div class="table sectionedit11"><table class="inline">
 
<tr class="row0">
 
<tr class="row0">
 
<th class="col0">amount</th><th class="col1">ingredient</th>
 
<th class="col0">amount</th><th class="col1">ingredient</th>
Line 282: Line 326:
 
</tr>
 
</tr>
 
</table></div>
 
</table></div>
<!-- EDIT12 TABLE [2225-2338] --><ul>
+
<!-- EDIT11 TABLE [2634-2747] --><ul>
 
<li class="level1"><div class="li"> incubation at 37°C for 1h</div>
 
<li class="level1"><div class="li"> incubation at 37°C for 1h</div>
 
</li>
 
</li>
Line 302: Line 346:
 
<strong>Digest: pOP and K592009 from clean-up with PstI (LS)</strong>
 
<strong>Digest: pOP and K592009 from clean-up with PstI (LS)</strong>
 
</p>
 
</p>
<div class="table sectionedit13"><table class="inline">
+
<div class="table sectionedit12"><table class="inline">
 
<tr class="row0">
 
<tr class="row0">
 
<th class="col0">amount</th><th class="col1">ingredient</th>
 
<th class="col0">amount</th><th class="col1">ingredient</th>
Line 319: Line 363:
 
</tr>
 
</tr>
 
</table></div>
 
</table></div>
<!-- EDIT13 TABLE [2550-2632] --><ul>
+
<!-- EDIT12 TABLE [2959-3041] --><ul>
 
<li class="level1"><div class="li"> incubation at 37°C, 1h</div>
 
<li class="level1"><div class="li"> incubation at 37°C, 1h</div>
 
</li>
 
</li>
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-nothing for K592009
 
-nothing for K592009
 
</p>
 
</p>
 +
<div class="thumb2 trien" style="width:310px"><div class="thumbinner"><a class="media" href="https://static.igem.org/mediawiki/2015/e/ed/Freiburg_labjournal-cloning-2015_09_05-1digestpop_k592009_ecoripsti.png" title="labjournal:cloning:2015_09_05-1digestpop_k592009_ecoripsti.png"><img alt="" class="mediabox2" src="https://static.igem.org/mediawiki/2015/e/ed/Freiburg_labjournal-cloning-2015_09_05-1digestpop_k592009_ecoripsti.png" width="300"/></a><div class="thumbcaption"><div class="magnify"><a class="internal" href="https://static.igem.org/mediawiki/2015/e/ed/Freiburg_labjournal-cloning-2015_09_05-1digestpop_k592009_ecoripsti.png" title="vergrößern"><img alt="" height="11" src="/igem2015/lib/plugins/imagebox/magnify-clip.png" width="15"/></a></div>Digest of pOP and K592009 ith EcoRI an dPstI. Expected badn lenght for pOP: ~5500bp and for K592009 ~700bp. Digest worked for pOP, but not for K592009.</div></div></div><hr/>
 
<p>
 
<p>
 
<strong>Inoculation: 3x 5ml LB-Cml wit K592009 (LS)</strong>
 
<strong>Inoculation: 3x 5ml LB-Cml wit K592009 (LS)</strong>
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</ul>
 
</ul>
 
</div>
 
</div>
<!-- EDIT11 SECTION "2015/09/05" [2068-2821] -->
+
<!-- EDIT10 SECTION "2015/09/05" [2477-3465] -->
<h3 class="sectionedit14">2015/09/06</h3>
+
<h3 class="sectionedit13">2015/09/06</h3>
 
<div class="level3">
 
<div class="level3">
 
<p>
 
<p>
Line 349: Line 394:
 
</li>
 
</li>
 
</ul>
 
</ul>
<div class="table sectionedit15"><table class="inline">
+
<div class="table sectionedit14"><table class="inline">
 
<tr class="row0">
 
<tr class="row0">
 
<td class="col0">1</td><td class="col1">77.0 ng/µl</td>
 
<td class="col0">1</td><td class="col1">77.0 ng/µl</td>
Line 360: Line 405:
 
</tr>
 
</tr>
 
</table></div>
 
</table></div>
<!-- EDIT15 TABLE [2916-2963] -->
+
<!-- EDIT14 TABLE [3560-3607] -->
 
<p>
 
<p>
 
<strong> Digest of all three preps with EcoRI (JN) </strong>
 
<strong> Digest of all three preps with EcoRI (JN) </strong>
 
</p>
 
</p>
<div class="table sectionedit16"><table class="inline">
+
<div class="table sectionedit15"><table class="inline">
 
<tr class="row0">
 
<tr class="row0">
 
<th class="col0">amount</th><th class="col1">ingredient</th>
 
<th class="col0">amount</th><th class="col1">ingredient</th>
Line 381: Line 426:
 
</tr>
 
</tr>
 
</table></div>
 
</table></div>
<!-- EDIT16 TABLE [3014-3091] --><ul>
+
<!-- EDIT15 TABLE [3658-3735] --><ul>
 
<li class="level1"><div class="li"> incubation at 37°C for 1h</div>
 
<li class="level1"><div class="li"> incubation at 37°C for 1h</div>
 +
</li>
 +
</ul>
 +
<p>
 +
<strong>PCR clean-up (JN)</strong>
 +
</p>
 +
<ul>
 +
<li class="level1"><div class="li"> Qiagen kit</div>
 +
</li>
 +
<li class="level1"><div class="li"> eluted in 20 µl dH2O</div>
 +
</li>
 +
</ul>
 +
<div class="table sectionedit16"><table class="inline">
 +
<tr class="row0">
 +
<td class="col0">1</td><td class="col1">7.6 ng/µl</td>
 +
</tr>
 +
<tr class="row1">
 +
<td class="col0">2</td><td class="col1">9.2 ng/µl</td>
 +
</tr>
 +
<tr class="row2">
 +
<td class="col0">3</td><td class="col1">7.2 ng/µl</td>
 +
</tr>
 +
</table></div>
 +
<!-- EDIT16 TABLE [3833-3877] -->
 +
<p>
 +
<strong>Digest: K592009 with PstI (JN)</strong>
 +
</p>
 +
<div class="table sectionedit17"><table class="inline">
 +
<tr class="row0">
 +
<th class="col0">amount</th><th class="col1">ingredient</th>
 +
</tr>
 +
<tr class="row1">
 +
<td class="col0">25µl</td><td class="col1">DNA</td>
 +
</tr>
 +
<tr class="row2">
 +
<td class="col0">1µl</td><td class="col1">PstI</td>
 +
</tr>
 +
<tr class="row3">
 +
<td class="col0">5µl</td><td class="col1">buffer 3.1</td>
 +
</tr>
 +
<tr class="row4">
 +
<td class="col0">19µl</td><td class="col1">dH20</td>
 +
</tr>
 +
</table></div>
 +
<!-- EDIT17 TABLE [3915-3989] --><ul>
 +
<li class="level1"><div class="li"> incubation at 37°C for 1h</div>
 +
</li>
 +
<li class="level1"><div class="li"> analysis on 1% agarose gel</div>
 +
</li>
 +
</ul>
 +
<p>
 +
<strong>Gel-ex: Digest of K592009 and pOP (LS)</strong>
 +
</p>
 +
<ul>
 +
<li class="level1"><div class="li"> qiagen kit</div>
 +
</li>
 +
<li class="level1"><div class="li"> eluted in 20µl</div>
 +
</li>
 +
<li class="level1"><div class="li"> pOP: 3,9 ng/µl</div>
 +
</li>
 +
<li class="level1"><div class="li"> K59209: 9,0 ng/µl</div>
 +
</li>
 +
</ul>
 +
<p>
 +
<strong>Ligation: K592009 into pOP (LS)</strong>
 +
</p>
 +
<div class="table sectionedit18"><table class="inline">
 +
<tr class="row0">
 +
<th class="col0">ingredient</th><th class="col1">amount [µl]</th>
 +
</tr>
 +
<tr class="row1">
 +
<td class="col0">pOP</td><td class="col1">12,83</td>
 +
</tr>
 +
<tr class="row2">
 +
<td class="col0">K592009</td><td class="col1">2,33</td>
 +
</tr>
 +
<tr class="row3">
 +
<td class="col0">T4 ligase buffer</td><td class="col1">2</td>
 +
</tr>
 +
<tr class="row4">
 +
<td class="col0">T4 ligase</td><td class="col1">1</td>
 +
</tr>
 +
<tr class="row5">
 +
<td class="col0">dH2o</td><td class="col1">2</td>
 +
</tr>
 +
</table></div>
 +
<!-- EDIT18 TABLE [4217-4313] --><ul>
 +
<li class="level1"><div class="li"> incubation at Rt for 1h</div>
 +
</li>
 +
</ul>
 +
<p>
 +
<strong>Transformation of ligation into <em>E.coli</em> T10 (LS)</strong>
 +
</p>
 +
<ul>
 +
<li class="level1"><div class="li"> 7µl of ligation mix</div>
 +
</li>
 +
<li class="level1"><div class="li"> transformation according to protocol</div>
 +
</li>
 +
<li class="level1"><div class="li"> plated on LB-Amp</div>
 +
</li>
 +
<li class="level1"><div class="li"> incubation at 37°C o/n</div>
 
</li>
 
</li>
 
</ul>
 
</ul>
 
</div>
 
</div>
<!-- EDIT14 SECTION "2015/09/06" [2822-] -->
+
<!-- EDIT13 SECTION "2015/09/06" [3466-4516] -->
 
+
<h3 class="sectionedit19">2015/09/10</h3>
 +
<div class="level3">
 +
<p>
 +
<strong> Clean-up of pOP EcoRI digest (JN) </strong>
 +
</p>
 +
<ul>
 +
<li class="level1"><div class="li"> Qiagen kit</div>
 +
</li>
 +
<li class="level1"><div class="li"> eluted in 20µl dH2O</div>
 +
</li>
 +
<li class="level1"><div class="li"> concentration: 1.8ng/µl</div>
 +
</li>
 +
</ul>
 +
<p>
 +
<strong> Subsequent digest with PstI (JN) </strong>
 +
</p>
 +
<div class="table sectionedit20"><table class="inline">
 +
<tr class="row0">
 +
<th class="col0"> ingredient</th><th class="col1"> amount [µl]</th>
 +
</tr>
 +
<tr class="row1">
 +
<td class="col0">DNA</td><td class="col1">16</td>
 +
</tr>
 +
<tr class="row2">
 +
<td class="col0">PstI</td><td class="col1">1</td>
 +
</tr>
 +
<tr class="row3">
 +
<td class="col0">buffer 3.1</td><td class="col1">5</td>
 +
</tr>
 +
<tr class="row4">
 +
<td class="col0">dH2O</td><td class="col1">28</td>
 +
</tr>
 +
</table></div>
 +
<!-- EDIT20 TABLE [4688-4758] --><ul>
 +
<li class="level1"><div class="li"> 37°C for 1h</div>
 +
</li>
 +
<li class="level1"><div class="li"> analysis on 1% agarose gel, see below</div>
 +
</li>
 +
</ul>
 +
<p>
 +
<strong> Digest of pOP_Anderson_GFP with EcoRI (JN) </strong>
 +
</p>
 +
<div class="table sectionedit21"><table class="inline">
 +
<tr class="row0">
 +
<th class="col0"> ingredient</th><th class="col1"> amount [µl]</th>
 +
</tr>
 +
<tr class="row1">
 +
<td class="col0">DNA</td><td class="col1">5</td>
 +
</tr>
 +
<tr class="row2">
 +
<td class="col0">EcoRI</td><td class="col1">1</td>
 +
</tr>
 +
<tr class="row3">
 +
<td class="col0">CutSmart</td><td class="col1">2</td>
 +
</tr>
 +
<tr class="row4">
 +
<td class="col0">dH2O</td><td class="col1">12</td>
 +
</tr>
 +
</table></div>
 +
<!-- EDIT21 TABLE [4870-4938] --><ul>
 +
<li class="level1"><div class="li"> 37°C for 1h</div>
 +
</li>
 +
</ul>
 +
<p>
 +
<strong> Clean-up of pOP_A_GFP EcoRI digest (JN) </strong>
 +
</p>
 +
<ul>
 +
<li class="level1"><div class="li"> Qiagen kit</div>
 +
</li>
 +
<li class="level1"><div class="li"> eluted in 20µl dH2O</div>
 +
</li>
 +
<li class="level1"><div class="li"> concentration: 28.0 ng/µl</div>
 +
</li>
 +
</ul>
 +
<p>
 +
<strong> Subsequent digest with PstI (JN) </strong>
 +
</p>
 +
<div class="table sectionedit22"><table class="inline">
 +
<tr class="row0">
 +
<th class="col0"> ingredient</th><th class="col1"> amount [µl]</th>
 +
</tr>
 +
<tr class="row1">
 +
<td class="col0">DNA</td><td class="col1">16</td>
 +
</tr>
 +
<tr class="row2">
 +
<td class="col0">PstI</td><td class="col1">1</td>
 +
</tr>
 +
<tr class="row3">
 +
<td class="col0">buffer 3.1</td><td class="col1">5</td>
 +
</tr>
 +
<tr class="row4">
 +
<td class="col0">dH2O</td><td class="col1">28</td>
 +
</tr>
 +
</table></div>
 +
<!-- EDIT22 TABLE [5116-5186] --><ul>
 +
<li class="level1"><div class="li"> 37°C for 1h</div>
 +
</li>
 +
<li class="level1"><div class="li"> analysis on 1% agarose gel</div>
 +
</li>
 +
</ul>
 +
<p>
 +
<strong> Gel-ex of pOP digested with EcoRI and PstI (JN) </strong>
 +
</p>
 +
<ul>
 +
<li class="level1"><div class="li"> Qiagen kit</div>
 +
</li>
 +
<li class="level1"><div class="li"> eluted in 20µl dH2O</div>
 +
</li>
 +
<li class="level1"><div class="li"> concentration: 9.6 ng/µl</div>
 +
</li>
 +
</ul>
 +
</div>
 +
<!-- EDIT19 SECTION "2015/09/10" [4517-5361] -->
 +
<h3 class="sectionedit23">2015/09/11</h3>
 +
<div class="level3">
 +
<p>
 +
<strong> Liquid cultures of Ligation pOP + BBa_K592009 (JN) </strong>
 +
</p>
 +
<ul>
 +
<li class="level1"><div class="li"> 10 colonies were picked</div>
 +
</li>
 +
<li class="level1"><div class="li"> inoculated in 5ml LB-Amp each</div>
 +
</li>
 +
<li class="level1"><div class="li"> 37°C, shaking, until evening</div>
 +
</li>
 +
</ul>
 +
<p>
 +
<strong> OD of liquid cultures (JN) </strong>
 +
</p>
 +
<ul>
 +
<li class="level1"><div class="li"> OD of the liquid cultures from the morning was measured</div>
 +
</li>
 +
<li class="level1"><div class="li"> cultures were diluted to a OD of 0.4 in a volume of 5ml and grown again for half an hour</div>
 +
</li>
 +
<li class="level1"><div class="li"> induction with 0.5µl of IPTG</div>
 +
</li>
 +
<li class="level1"><div class="li"> incubated o/n shaking at 37°C</div>
 +
</li>
 +
</ul>
 +
</div>
 +
<!-- EDIT23 SECTION "2015/09/11" [5362-5792] -->
 +
<h3 class="sectionedit24">2015/09/12</h3>
 +
<div class="level3">
 +
<p>
 +
<strong> Mini-Prep of 3 induced cultures of pOP_K592009 (JN) </strong>
 +
</p>
 +
<ul>
 +
<li class="level1"><div class="li"> unfortunately, no liquid were blue as they should be if expressing the protein</div>
 +
</li>
 +
<li class="level1"><div class="li"> Zymo Research kit</div>
 +
</li>
 +
<li class="level1"><div class="li"> eluted in 30µl dH2O</div>
 +
</li>
 +
</ul>
 +
<div class="table sectionedit25"><table class="inline">
 +
<tr class="row0">
 +
<th class="col0"> </th><th class="col1"> concentration [ng/µl]</th>
 +
</tr>
 +
<tr class="row1">
 +
<td class="col0">pOP_K592009 1</td><td class="col1">48.7</td>
 +
</tr>
 +
<tr class="row2">
 +
<td class="col0">pOP_K592009 4</td><td class="col1">46.5</td>
 +
</tr>
 +
<tr class="row3">
 +
<td class="col0">pOP_K592009 7</td><td class="col1">52.7</td>
 +
</tr>
 +
</table></div>
 +
<!-- EDIT25 TABLE [6003-6093] -->
 +
<p>
 +
<strong> Test-digest of pOP_K592009 with EcoRI (JN) </strong>
 +
</p>
 +
<div class="table sectionedit26"><table class="inline">
 +
<tr class="row0">
 +
<th class="col0"> ingredient</th><th class="col1"> amount [µl]</th>
 +
</tr>
 +
<tr class="row1">
 +
<td class="col0">DNA</td><td class="col1">15</td>
 +
</tr>
 +
<tr class="row2">
 +
<td class="col0">EcoRI</td><td class="col1">1</td>
 +
</tr>
 +
<tr class="row3">
 +
<td class="col0">CutSmart</td><td class="col1">2</td>
 +
</tr>
 +
<tr class="row4">
 +
<td class="col0">dH2O</td><td class="col1">2</td>
 +
</tr>
 +
</table></div>
 +
<!-- EDIT26 TABLE [6145-6213] --><ul>
 +
<li class="level1"><div class="li"> 37°C for 1h</div>
 +
</li>
 +
</ul>
 +
<p>
 +
<strong> Clean-up of pOP_K592009 EcoRI digest (JN) </strong>
 +
</p>
 +
<ul>
 +
<li class="level1"><div class="li"> Qiagen kit</div>
 +
</li>
 +
<li class="level1"><div class="li"> eluted in 20µl dH2O</div>
 +
</li>
 +
</ul>
 +
<div class="table sectionedit27"><table class="inline">
 +
<tr class="row0">
 +
<th class="col0"> </th><th class="col1"> concentration [ng/µl]</th>
 +
</tr>
 +
<tr class="row1">
 +
<td class="col0">pOP_K592009 1</td><td class="col1">20.0</td>
 +
</tr>
 +
<tr class="row2">
 +
<td class="col0">pOP_K592009 4</td><td class="col1">14.0</td>
 +
</tr>
 +
<tr class="row3">
 +
<td class="col0">pOP_K592009 7</td><td class="col1">13.7</td>
 +
</tr>
 +
</table></div>
 +
<!-- EDIT27 TABLE [6322-6412] -->
 +
<p>
 +
<strong> Subsequent digest with PstI (JN) </strong>
 +
</p>
 +
<div class="table sectionedit28"><table class="inline">
 +
<tr class="row0">
 +
<th class="col0"> ingredient</th><th class="col1"> amount [µl]</th>
 +
</tr>
 +
<tr class="row1">
 +
<td class="col0">DNA</td><td class="col1">16</td>
 +
</tr>
 +
<tr class="row2">
 +
<td class="col0">PstI</td><td class="col1">1</td>
 +
</tr>
 +
<tr class="row3">
 +
<td class="col0">buffer 3.1</td><td class="col1">2</td>
 +
</tr>
 +
<tr class="row4">
 +
<td class="col0">dH2O</td><td class="col1">1</td>
 +
</tr>
 +
</table></div>
 +
<!-- EDIT28 TABLE [6454-6523] --><ul>
 +
<li class="level1"><div class="li"> 37°C for 1h</div>
 +
</li>
 +
<li class="level1"><div class="li"> analysis on 1% agarose gel, band fot the backbone was clearly visible at the right height but no band for the insert was visible</div>
 +
</li>
 +
</ul>
 +
<p>
 +
<strong>Digest: BBa_I13504 with EcoRI (LS)</strong>
 +
</p>
 +
<div class="table sectionedit29"><table class="inline">
 +
<tr class="row0">
 +
<th class="col0">ingredients</th><th class="col1">volume</th>
 +
</tr>
 +
<tr class="row1">
 +
<td class="col0">BBa_I13504</td><td class="col1">10µl</td>
 +
</tr>
 +
<tr class="row2">
 +
<td class="col0">CutSmart</td><td class="col1">4µl</td>
 +
</tr>
 +
<tr class="row3">
 +
<td class="col0">EcoRI</td><td class="col1">1µl</td>
 +
</tr>
 +
<tr class="row4">
 +
<td class="col0">dH2O</td><td class="col1">25µl</td>
 +
</tr>
 +
</table></div>
 +
<!-- EDIT29 TABLE [6717-6798] --><ul>
 +
<li class="level1"><div class="li"> incubation at 37°C for 1h</div>
 +
</li>
 +
<li class="level1"><div class="li"> clean-up with PCR-Clean-up kit (Roche)</div>
 +
</li>
 +
<li class="level1"><div class="li"> eluted in 20µl: 34,5ng/µl</div>
 +
</li>
 +
</ul>
 +
<p>
 +
<strong>Digest: Clean-up (BBa_I13504) with PstI (LS)</strong>
 +
</p>
 +
<div class="table sectionedit30"><table class="inline">
 +
<tr class="row0">
 +
<th class="col0">ingredients</th><th class="col1">volumes</th>
 +
</tr>
 +
<tr class="row1">
 +
<td class="col0">BBa_I13504 (EcoRI digested)</td><td class="col1"></td>
 +
</tr>
 +
<tr class="row2">
 +
<td class="col0">buffer 3.1</td><td class="col1">4µl</td>
 +
</tr>
 +
<tr class="row3">
 +
<td class="col0">PstI</td><td class="col1">1µl</td>
 +
</tr>
 +
<tr class="row4">
 +
<td class="col0">dH20</td><td class="col1">17µl</td>
 +
</tr>
 +
</table></div>
 +
<!-- EDIT30 TABLE [6957-7057] --><ul>
 +
<li class="level1"><div class="li"> incubation at 37°C for 1h</div>
 +
</li>
 +
<li class="level1"><div class="li"> analysis on 1% agarose gel, correct band of the BioBrick was cut out of the gel</div>
 +
</li>
 +
</ul>
 +
<p>
 +
<strong> Gel-ex of BBa_I13504 (JN) </strong>
 +
</p>
 +
<ul>
 +
<li class="level1"><div class="li"> Qiagen kit</div>
 +
</li>
 +
<li class="level1"><div class="li"> eluted in 20µl dH2O</div>
 +
</li>
 +
<li class="level1"><div class="li"> concentration: 9.1ng/µl</div>
 +
</li>
 +
</ul>
 +
<p>
 +
<strong> Ligation of pOP + BBa_I13504 (JN) </strong>
 +
</p>
 +
<div class="table sectionedit31"><table class="inline">
 +
<tr class="row0">
 +
<th class="col0">ingredients</th><th class="col1">volumes</th>
 +
</tr>
 +
<tr class="row1">
 +
<td class="col0">pOP</td><td class="col1">7µl</td>
 +
</tr>
 +
<tr class="row2">
 +
<td class="col0">BBa_I13504</td><td class="col1">2.9µl</td>
 +
</tr>
 +
<tr class="row3">
 +
<td class="col0">buffer</td><td class="col1">2µl</td>
 +
</tr>
 +
<tr class="row4">
 +
<td class="col0">T4ligase</td><td class="col1">1µl</td>
 +
</tr>
 +
<tr class="row5">
 +
<td class="col0">dH20</td><td class="col1">7.1µl</td>
 +
</tr>
 +
</table></div>
 +
<!-- EDIT31 TABLE [7318-7414] --><ul>
 +
<li class="level1"><div class="li"> 1h at RT</div>
 +
</li>
 +
</ul>
 +
<p>
 +
<strong> Trafo of pOP_I13504 into BL21 (LS) </strong>
 +
</p>
 +
<ul>
 +
<li class="level1"><div class="li"> 7 µl of ligation mix</div>
 +
</li>
 +
<li class="level1"><div class="li"> transformation according to protocol</div>
 +
</li>
 +
<li class="level1"><div class="li"> plated on LB-Amp</div>
 +
</li>
 +
</ul>
 +
</div>
 +
<!-- EDIT24 SECTION "2015/09/12" [5793-7560] -->
 +
<h3 class="sectionedit32">2015/09/13</h3>
 +
<div class="level3">
 +
<p>
 +
<strong> Inoculation of pOP_I13504 (LS) </strong>
 +
</p>
 +
<ul>
 +
<li class="level1"><div class="li"> inoculation of 10 x 5 ml LB-Amp with  ligation clones</div>
 +
</li>
 +
<li class="level1"><div class="li"> incubation at 37°C</div>
 +
</li>
 +
</ul>
 +
<p>
 +
<strong> OD Measurement of liquid cultures (JN) </strong>
 +
</p>
 +
<ul>
 +
<li class="level1"><div class="li"> OD for each liquid culture was measured</div>
 +
</li>
 +
<li class="level1"><div class="li"> dilution for each culture to a final OD of 0.4</div>
 +
</li>
 +
<li class="level1"><div class="li"> incubation at 37°C for 30min, shaking</div>
 +
</li>
 +
<li class="level1"><div class="li"> OD was measured again –&gt; for all cultures about 0.5</div>
 +
</li>
 +
<li class="level1"><div class="li"> induction with 5µl IPTG for a final concentration of 1mM</div>
 +
</li>
 +
<li class="level1"><div class="li"> incubation at 37°C, shaking</div>
 +
</li>
 +
</ul>
 +
<p>
 +
<strong>Checked for fluorescence (JN/LS)</strong>
 +
</p>
 +
<ul>
 +
<li class="level1"><div class="li"> measuring of fluorescence in all 10 cultures with UV light</div>
 +
</li>
 +
<li class="level1"><div class="li"> all cultures showed fluorescence</div>
 +
</li>
 +
</ul>
 +
<p>
 +
<strong>Restreaked 3 cultures of pOP_I13504 (LS)</strong>
 +
</p>
 +
<ul>
 +
<li class="level1"><div class="li"> incubation at 37°C o/n</div>
 +
</li>
 +
</ul>
 +
</div>
 +
<!-- EDIT32 SECTION "2015/09/13" [7561-8254] -->
 +
<h3 class="sectionedit33">2015/09/14</h3>
 +
<div class="level3">
 +
<p>
 +
<strong>Inoculation of pOP_I13504 (LS)</strong>
 +
</p>
 +
<ul>
 +
<li class="level1"><div class="li"> 6 x 5ml LB-Amp, two cultures of each colony</div>
 +
</li>
 +
<li class="level1"><div class="li"> incubation at 37°C, shaking</div>
 +
</li>
 +
</ul>
 +
<p>
 +
<strong>OD measurement + induction with IPTG o/n (JN)</strong>
 +
</p>
 +
<ul>
 +
<li class="level1"><div class="li"> cultures were diluted to obtain an OD of 0.3</div>
 +
</li>
 +
<li class="level1"><div class="li"> incubation for ca. half an hour shaking at 37°C</div>
 +
</li>
 +
<li class="level1"><div class="li"> OD measurement until OD of approximately 0.5 was reached</div>
 +
</li>
 +
<li class="level1"><div class="li"> one culture of each colony was induced with IPTG in a final concentration of 1 mM, the other was left uninduced</div>
 +
</li>
 +
<li class="level1"><div class="li"> incubation o/n at 30°C, shaking</div>
 +
</li>
 +
</ul>
 +
</div>
 +
<!-- EDIT33 SECTION "2015/09/14" [8255-8761] -->
 +
<h3 class="sectionedit34">2015/09/15</h3>
 +
<div class="level3">
 +
<p>
 +
<strong>GFP measurement with UV light (JN)</strong>
 +
</p>
 +
<ul>
 +
<li class="level1"><div class="li"> expressed GFP was visible with the bare eye as well as under UV light</div>
 +
</li>
 +
<li class="level1"><div class="li"> one liquid culture was pelleted, 1 ml for following SDS-PAGE, the rest to be prepped</div>
 +
</li>
 +
</ul>
 +
<div class="thumb2 trien" style="width:310px"><div class="thumbinner"><a class="media" href="https://static.igem.org/mediawiki/2015/e/e8/Freiburg_labjournal-cloning-2015_09_15_pop_gfp_pellet.jpg" title="labjournal:cloning:2015_09_15_pop_gfp_pellet.jpg"><img alt="" class="mediabox2" src="https://static.igem.org/mediawiki/2015/e/e8/Freiburg_labjournal-cloning-2015_09_15_pop_gfp_pellet.jpg" width="300"/></a><div class="thumbcaption"><div class="magnify"><a class="internal" href="https://static.igem.org/mediawiki/2015/e/e8/Freiburg_labjournal-cloning-2015_09_15_pop_gfp_pellet.jpg" title="vergrößern"><img alt="" height="11" src="/igem2015/lib/plugins/imagebox/magnify-clip.png" width="15"/></a></div>Liquid culture pellet of the same colony. One culture was induced with IPTG, the other not.</div></div></div><hr/>
 +
<p>
 +
<strong>SDS-PAGE (Protein Purification Labjournal JD)</strong>
 +
</p>
 +
<ul>
 +
<li class="level1"><div class="li"> 1 ml of liquid culture was pelleted and the supernatant discarded</div>
 +
</li>
 +
<li class="level1"><div class="li"> 300 µl of 2.5x SDS Loading-dye was added</div>
 +
</li>
 +
<li class="level1"><div class="li"> boiling at 95°C for 10min, cooldown</div>
 +
</li>
 +
<li class="level1"><div class="li"> 20 µl were loaded on a 12.5% SDS-PAGE gel</div>
 +
</li>
 +
<li class="level1"><div class="li"> analysis via coomassie staining</div>
 +
</li>
 +
</ul>
 +
<div class="thumb2 trien" style="width:310px"><div class="thumbinner"><a class="media" href="https://static.igem.org/mediawiki/2015/4/4a/Freiburg_labjournal-cloning-2015_09_15_pop_gfp_sds.jpg" title="labjournal:cloning:2015_09_15_pop_gfp_sds.jpg"><img alt="" class="mediabox2" src="https://static.igem.org/mediawiki/2015/4/4a/Freiburg_labjournal-cloning-2015_09_15_pop_gfp_sds.jpg" width="300"/></a><div class="thumbcaption"><div class="magnify"><a class="internal" href="https://static.igem.org/mediawiki/2015/4/4a/Freiburg_labjournal-cloning-2015_09_15_pop_gfp_sds.jpg" title="vergrößern"><img alt="" height="11" src="/igem2015/lib/plugins/imagebox/magnify-clip.png" width="15"/></a></div>SDS-PAGE of pOP, enrichment of expressed GFP (27 kDa) visible from the induced culture</div></div></div>
 +
</div>
 +
<!-- EDIT34 SECTION "2015/09/15" [8762-] -->
 +
</div>
 
</html>
 
</html>
 
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{{Freiburg/wiki_content_end}}
 
{{Freiburg/wiki_content_end}}

Latest revision as of 07:17, 20 November 2015

""

2015/09/02

Digest: pOP, pILS3 and K592009 with EcoRI (LS)

amountingredient
~5µlplasmid
5µlCut Smart
1µlEcoRI
up to 50µldH2O
  • incubation at 37°C, ~1h

PCR Purification of EcoRI digest (JN)

  • Qiagen kit
  • eluted in 20µl dH2O
concentration [ng/µl]
pOP 3.3
pILS3 23.
BBa_K592009 2.3

Digest of pOP, pILS3 and BBa_K592009 with PstI (JN)

ingredient amount [µl]
DNA16/17/18
PstI1
buffer 3.15
dH2O28/27/26
  • 37°C for 1h
  • analysis on 1% agarose gel
Expected fragment sizes: pOP ~5000bp, BBa_I13504 ~700bp, BBa_K592009 ~700bp

Gel-ex of BBa_I13504 (JN)

  • Qiagen kit
  • eluted in 20µl dH2O
  • concentration: 10.2ng/µl

Repetition of pOP Digest with EcoRI (JN)

ingredient amount [µl]
DNA5
EcoRI1
CutSmart5
dH2O39
  • 37°C for 1h

PCR Purification of pOP Digest

  • Qiagen kit
  • eluted in 20µl dH20
  • concentration: 3.7ng/µl

pOP digest with PstI

ingredient amount [µl]
DNA17
PstI1
buffer 3.15
dH2O27
  • 37°C for 1h
  • analysis on 1% agarose gel
Expected fragment size: ~5000bp

Gel-ex of pOP (JN)

  • Qiagen kit
  • eluted in 20µl dH2O
  • concentration: 6.4ng/µl

Ligation of pOP+BBa_I13504 (JN)

  • Backbone: 3.4 µl
  • BBa_I13504: 10.5 µl
  • buffer: 2 µl
  • T4 ligase: 1 µl
  • dH2O: 3.1 µl

Trafo: Ligation of pOP BBA_I13504 in E.coli T10 (LS)

  • 7 µl of ligation
  • transformation according to protocol

2015/09/03

Inoculation of pOP_BBa_I13504 (LS)

  • picked 3 clones
  • inoculation in LB-Amp (5ml)
  • incubation at 37°C

Inoculation of pOP_BBa_I13504 (JN)

  • 10 more clones were picked and inoculated o/n
  • each clone in 5ml of LB-Amp

2015/09/04

Mini-Prep of pOP_Anderson_GFP (JN)

  • all liquid cultures were fluorescent, only three were prepped, four more were pelleted in stored in the freezer as backup
  • Qiagen kit
  • eluted in 30µl dH2O
concentration [ng/µl]
pOP_Anderson_GFP 4 21.1
pOP_Anderson_GFP 9 42.9
pOP_Anderson_GFP 11 37.2
  • sample 9 and 11 were sent in for sequencing

Inoculation of pOP (JN)

  • inoculation of 3 colonies from already performed re-trafo (LS)
  • 3 liquid cultures with 5ml LB-Amp medium each
  • inoculated o/n

2015/09/05

Miniprep: pOP (LS)

  • 15µl of pOP
  • Zymo kit
  • elution in 65µl: 81,0 ng/µl

Digest: pOP and K592009 with EcoRI (LS)

amountingredient
10µl/~4µlpOP/K592009(all that was left)
1µlEcoRI
5µlCut Smart
up to 50µldH20
  • incubation at 37°C for 1h

Clean-up of Digest (LS)

  • Zymo PCR clean-up kit
  • elution in 20µl:
  • pOP:21,3ng/µl
  • K592009:2,7ng/µl

Digest: pOP and K592009 from clean-up with PstI (LS)

amountingredient
18µlpOP/K592009
5µlbuffer 3.1
1µlPstI
36µl dH2O
  • incubation at 37°C, 1h
  • analysis on 1% agarose gel:

-small band visible for pOP -nothing for K592009

Digest of pOP and K592009 ith EcoRI an dPstI. Expected badn lenght for pOP: ~5500bp and for K592009 ~700bp. Digest worked for pOP, but not for K592009.

Inoculation: 3x 5ml LB-Cml wit K592009 (LS)

  • incubation at 37°C, o/n

2015/09/06

Mini-Prep of K592009 (JN)

  • Qiagen kit
  • eluted in 20µl dH2O
177.0 ng/µl
294.4 ng/µl
364.6 ng/µl

Digest of all three preps with EcoRI (JN)

amountingredient
5µlK592009
1µlEcoRI
5µlCut Smart
39µldH20
  • incubation at 37°C for 1h

PCR clean-up (JN)

  • Qiagen kit
  • eluted in 20 µl dH2O
17.6 ng/µl
29.2 ng/µl
37.2 ng/µl

Digest: K592009 with PstI (JN)

amountingredient
25µlDNA
1µlPstI
5µlbuffer 3.1
19µldH20
  • incubation at 37°C for 1h
  • analysis on 1% agarose gel

Gel-ex: Digest of K592009 and pOP (LS)

  • qiagen kit
  • eluted in 20µl
  • pOP: 3,9 ng/µl
  • K59209: 9,0 ng/µl

Ligation: K592009 into pOP (LS)

ingredientamount [µl]
pOP12,83
K5920092,33
T4 ligase buffer2
T4 ligase1
dH2o2
  • incubation at Rt for 1h

Transformation of ligation into E.coli T10 (LS)

  • 7µl of ligation mix
  • transformation according to protocol
  • plated on LB-Amp
  • incubation at 37°C o/n

2015/09/10

Clean-up of pOP EcoRI digest (JN)

  • Qiagen kit
  • eluted in 20µl dH2O
  • concentration: 1.8ng/µl

Subsequent digest with PstI (JN)

ingredient amount [µl]
DNA16
PstI1
buffer 3.15
dH2O28
  • 37°C for 1h
  • analysis on 1% agarose gel, see below

Digest of pOP_Anderson_GFP with EcoRI (JN)

ingredient amount [µl]
DNA5
EcoRI1
CutSmart2
dH2O12
  • 37°C for 1h

Clean-up of pOP_A_GFP EcoRI digest (JN)

  • Qiagen kit
  • eluted in 20µl dH2O
  • concentration: 28.0 ng/µl

Subsequent digest with PstI (JN)

ingredient amount [µl]
DNA16
PstI1
buffer 3.15
dH2O28
  • 37°C for 1h
  • analysis on 1% agarose gel

Gel-ex of pOP digested with EcoRI and PstI (JN)

  • Qiagen kit
  • eluted in 20µl dH2O
  • concentration: 9.6 ng/µl

2015/09/11

Liquid cultures of Ligation pOP + BBa_K592009 (JN)

  • 10 colonies were picked
  • inoculated in 5ml LB-Amp each
  • 37°C, shaking, until evening

OD of liquid cultures (JN)

  • OD of the liquid cultures from the morning was measured
  • cultures were diluted to a OD of 0.4 in a volume of 5ml and grown again for half an hour
  • induction with 0.5µl of IPTG
  • incubated o/n shaking at 37°C

2015/09/12

Mini-Prep of 3 induced cultures of pOP_K592009 (JN)

  • unfortunately, no liquid were blue as they should be if expressing the protein
  • Zymo Research kit
  • eluted in 30µl dH2O
concentration [ng/µl]
pOP_K592009 148.7
pOP_K592009 446.5
pOP_K592009 752.7

Test-digest of pOP_K592009 with EcoRI (JN)

ingredient amount [µl]
DNA15
EcoRI1
CutSmart2
dH2O2
  • 37°C for 1h

Clean-up of pOP_K592009 EcoRI digest (JN)

  • Qiagen kit
  • eluted in 20µl dH2O
concentration [ng/µl]
pOP_K592009 120.0
pOP_K592009 414.0
pOP_K592009 713.7

Subsequent digest with PstI (JN)

ingredient amount [µl]
DNA16
PstI1
buffer 3.12
dH2O1
  • 37°C for 1h
  • analysis on 1% agarose gel, band fot the backbone was clearly visible at the right height but no band for the insert was visible

Digest: BBa_I13504 with EcoRI (LS)

ingredientsvolume
BBa_I1350410µl
CutSmart4µl
EcoRI1µl
dH2O25µl
  • incubation at 37°C for 1h
  • clean-up with PCR-Clean-up kit (Roche)
  • eluted in 20µl: 34,5ng/µl

Digest: Clean-up (BBa_I13504) with PstI (LS)

ingredientsvolumes
BBa_I13504 (EcoRI digested)
buffer 3.14µl
PstI1µl
dH2017µl
  • incubation at 37°C for 1h
  • analysis on 1% agarose gel, correct band of the BioBrick was cut out of the gel

Gel-ex of BBa_I13504 (JN)

  • Qiagen kit
  • eluted in 20µl dH2O
  • concentration: 9.1ng/µl

Ligation of pOP + BBa_I13504 (JN)

ingredientsvolumes
pOP7µl
BBa_I135042.9µl
buffer2µl
T4ligase1µl
dH207.1µl
  • 1h at RT

Trafo of pOP_I13504 into BL21 (LS)

  • 7 µl of ligation mix
  • transformation according to protocol
  • plated on LB-Amp

2015/09/13

Inoculation of pOP_I13504 (LS)

  • inoculation of 10 x 5 ml LB-Amp with ligation clones
  • incubation at 37°C

OD Measurement of liquid cultures (JN)

  • OD for each liquid culture was measured
  • dilution for each culture to a final OD of 0.4
  • incubation at 37°C for 30min, shaking
  • OD was measured again –> for all cultures about 0.5
  • induction with 5µl IPTG for a final concentration of 1mM
  • incubation at 37°C, shaking

Checked for fluorescence (JN/LS)

  • measuring of fluorescence in all 10 cultures with UV light
  • all cultures showed fluorescence

Restreaked 3 cultures of pOP_I13504 (LS)

  • incubation at 37°C o/n

2015/09/14

Inoculation of pOP_I13504 (LS)

  • 6 x 5ml LB-Amp, two cultures of each colony
  • incubation at 37°C, shaking

OD measurement + induction with IPTG o/n (JN)

  • cultures were diluted to obtain an OD of 0.3
  • incubation for ca. half an hour shaking at 37°C
  • OD measurement until OD of approximately 0.5 was reached
  • one culture of each colony was induced with IPTG in a final concentration of 1 mM, the other was left uninduced
  • incubation o/n at 30°C, shaking

2015/09/15

GFP measurement with UV light (JN)

  • expressed GFP was visible with the bare eye as well as under UV light
  • one liquid culture was pelleted, 1 ml for following SDS-PAGE, the rest to be prepped
Liquid culture pellet of the same colony. One culture was induced with IPTG, the other not.

SDS-PAGE (Protein Purification Labjournal JD)

  • 1 ml of liquid culture was pelleted and the supernatant discarded
  • 300 µl of 2.5x SDS Loading-dye was added
  • boiling at 95°C for 10min, cooldown
  • 20 µl were loaded on a 12.5% SDS-PAGE gel
  • analysis via coomassie staining
SDS-PAGE of pOP, enrichment of expressed GFP (27 kDa) visible from the induced culture