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

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<!-- Labjournal content goes in here -->
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<h3 class="sectionedit1">2015/09/01</h3>
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<div class="level3">
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</div>
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<!-- EDIT1 SECTION "2015/09/01" [1-22] -->
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<h3 class="sectionedit2">2015/09/02</h3>
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<div class="level3">
 +
<p>
 +
<strong>Digest: pOP, pILS3 and K592009 with EcoRI (LS)</strong>
 +
</p>
 +
<div class="table sectionedit3"><table class="inline">
 +
<tr class="row0">
 +
<th class="col0">amount</th><th class="col1">ingredient</th>
 +
</tr>
 +
<tr class="row1">
 +
<td class="col0">~5µl</td><td class="col1">plasmid</td>
 +
</tr>
 +
<tr class="row2">
 +
<td class="col0">5µl</td><td class="col1">Cut Smart</td>
 +
</tr>
 +
<tr class="row3">
 +
<td class="col0">1µl</td><td class="col1">EcoRI</td>
 +
</tr>
 +
<tr class="row4">
 +
<td class="col0">up to 50µl</td><td class="col1">dH2O</td>
 +
</tr>
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</table></div>
 +
<!-- EDIT3 TABLE [96-180] --><ul>
 +
<li class="level1"><div class="li"> incubation at 37°C, ~1h</div>
 +
</li>
 +
</ul>
 +
<p>
 +
<strong> PCR Purification of 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 sectionedit4"><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</td><td class="col1"> 3.3</td>
 +
</tr>
 +
<tr class="row2">
 +
<td class="col0">pILS3</td><td class="col1"> 23.</td>
 +
</tr>
 +
<tr class="row3">
 +
<td class="col0">BBa_K592009</td><td class="col1"> 2.3</td>
 +
</tr>
 +
</table></div>
 +
<!-- EDIT4 TABLE [298-368] -->
 +
<p>
 +
<strong> Digest of pOP, pILS3 and BBa_K592009 with PstI (JN) </strong>
 +
</p>
 +
<div class="table sectionedit5"><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">16/17/18</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">28/27/26</td>
 +
</tr>
 +
</table></div>
 +
<!-- EDIT5 TABLE [428-510] --><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 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: 10.2ng/µl</div>
 +
</li>
 +
</ul>
 +
<p>
 +
<strong> Repetition of pOP Digest with EcoRI (JN) </strong>
 +
</p>
 +
<div class="table sectionedit6"><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">5</td>
 +
</tr>
 +
<tr class="row4">
 +
<td class="col0">dH2O</td><td class="col1">39</td>
 +
</tr>
 +
</table></div>
 +
<!-- EDIT6 TABLE [712-780] --><ul>
 +
<li class="level1"><div class="li"> 37°C for 1h</div>
 +
</li>
 +
</ul>
 +
<p>
 +
<strong> PCR Purification of pOP Digest </strong>
 +
</p>
 +
<ul>
 +
<li class="level1"><div class="li"> Qiagen kit</div>
 +
</li>
 +
<li class="level1"><div class="li"> eluted in 20µl dH20</div>
 +
</li>
 +
<li class="level1"><div class="li"> concentration: 3.7ng/µl</div>
 +
</li>
 +
</ul>
 +
<p>
 +
<strong> pOP digest with PstI </strong>
 +
</p>
 +
<div class="table sectionedit7"><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">17</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">27</td>
 +
</tr>
 +
</table></div>
 +
<!-- EDIT7 TABLE [934-1004] --><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 (JN) </strong>
 +
</p>
 +
<p>
 +
<strong> Ligation of pOP+BBa_I13504 (JN) </strong>
 +
</p>
 +
<p>
 +
<strong> Trafo: Ligation of pOP BBA_I13504 in <em>E.coli</em> T10 (LS) </strong>
 +
</p>
 +
<ul>
 +
<li class="level1"><div class="li"> 10µl of ligation</div>
 +
</li>
 +
<li class="level1"><div class="li"> transformation according to protocol</div>
 +
</li>
 +
</ul>
 +
</div>
 +
<!-- EDIT2 SECTION "2015/09/02" [23-1246] -->
 +
<h3 class="sectionedit8">2015/09/03</h3>
 +
<div class="level3">
 +
<p>
 +
<strong> Inoculation of pOP_BBa_I13504 (LS) </strong>
 +
</p>
 +
<ul>
 +
<li class="level1"><div class="li"> picked 3 clones </div>
 +
</li>
 +
<li class="level1"><div class="li"> inoculation in LB-Amp (5ml)</div>
 +
</li>
 +
<li class="level1"><div class="li"> incubation at 37°C</div>
 +
</li>
 +
</ul>
 +
<p>
 +
<strong> Inoculation of pOP_BBa_I13504 (JN) </strong>
 +
</p>
 +
<ul>
 +
<li class="level1"><div class="li"> 10 more clones were picked and inoculated o/n</div>
 +
</li>
 +
<li class="level1"><div class="li"> each clone in 5ml of LB-Amp</div>
 +
</li>
 +
</ul>
 +
</div>
 +
<!-- EDIT8 SECTION "2015/09/03" [1247-1511] -->
 +
<h3 class="sectionedit9">2015/09/04</h3>
 +
<div class="level3">
 +
<p>
 +
<strong> Mini-Prep of pOP_Anderson_GFP (JN) </strong>
 +
</p>
 +
<ul>
 +
<li class="level1"><div class="li"> all liquid cultures were fluorescent, only three were prepped, four more were pelleted in stored in the freezer as backup</div>
 +
</li>
 +
<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 sectionedit10"><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_Anderson_GFP 4</td><td class="col1"> 21.1</td>
 +
</tr>
 +
<tr class="row2">
 +
<td class="col0">pOP_Anderson_GFP 9</td><td class="col1"> 42.9</td>
 +
</tr>
 +
<tr class="row3">
 +
<td class="col0">pOP_Anderson_GFP 11</td><td class="col1"> 37.2</td>
 +
</tr>
 +
</table></div>
 +
<!-- EDIT10 TABLE [1741-1850] --><ul>
 +
<li class="level1"><div class="li"> sample 9 and 11 were sent in for sequencing</div>
 +
</li>
 +
</ul>
 +
<p>
 +
<strong> Inoculation of pOP (JN) </strong>
 +
</p>
 +
<ul>
 +
<li class="level1"><div class="li"> inoculation of 3 colonies from already performed re-trafo (LS)</div>
 +
</li>
 +
<li class="level1"><div class="li"> 3 liquid cultures with 5ml LB-Amp medium each</div>
 +
</li>
 +
<li class="level1"><div class="li"> inoculated o/n</div>
 +
</li>
 +
</ul>
 +
</div>
 +
<!-- EDIT9 SECTION "2015/09/04" [1512-2067] -->
 +
<h3 class="sectionedit11">2015/09/05</h3>
 +
<div class="level3">
 +
<p>
 +
<strong>Miniprep: pOP (LS)</strong>
 +
</p>
 +
<ul>
 +
<li class="level1"><div class="li"> 15µl of pOP</div>
 +
</li>
 +
<li class="level1"><div class="li"> Zymo kit</div>
 +
</li>
 +
<li class="level1"><div class="li"> elution in 65µl: 81,0 ng/µl</div>
 +
</li>
 +
</ul>
 +
<p>
 +
<strong>Digest: pOP and K592009 with EcoRI (LS)</strong>
 +
</p>
 +
<div class="table sectionedit12"><table class="inline">
 +
<tr class="row0">
 +
<th class="col0">amount</th><th class="col1">ingredient</th>
 +
</tr>
 +
<tr class="row1">
 +
<td class="col0">10µl/~4µl</td><td class="col1">pOP/K592009(all that was left)</td>
 +
</tr>
 +
<tr class="row2">
 +
<td class="col0">1µl</td><td class="col1">EcoRI</td>
 +
</tr>
 +
<tr class="row3">
 +
<td class="col0">5µl</td><td class="col1">Cut Smart</td>
 +
</tr>
 +
<tr class="row4">
 +
<td class="col0">up to 50µl</td><td class="col1">dH20</td>
 +
</tr>
 +
</table></div>
 +
<!-- EDIT12 TABLE [2225-2338] --><ul>
 +
<li class="level1"><div class="li"> incubation at 37°C for 1h</div>
 +
</li>
 +
</ul>
 +
<p>
 +
<strong>Clean-up of Digest (LS)</strong>
 +
</p>
 +
<ul>
 +
<li class="level1"><div class="li"> Zymo PCR clean-up kit</div>
 +
</li>
 +
<li class="level1"><div class="li"> elution in 20µl:</div>
 +
</li>
 +
<li class="level1"><div class="li"> pOP:21,3ng/µl</div>
 +
</li>
 +
<li class="level1"><div class="li"> K592009:2,7ng/µl</div>
 +
</li>
 +
</ul>
 +
<p>
 +
<strong>Digest: pOP and K592009 from clean-up with PstI (LS)</strong>
 +
</p>
 +
<div class="table sectionedit13"><table class="inline">
 +
<tr class="row0">
 +
<th class="col0">amount</th><th class="col1">ingredient</th>
 +
</tr>
 +
<tr class="row1">
 +
<td class="col0">18µl</td><td class="col1">pOP/K592009</td>
 +
</tr>
 +
<tr class="row2">
 +
<td class="col0">5µl</td><td class="col1">buffer 3.1</td>
 +
</tr>
 +
<tr class="row3">
 +
<td class="col0">1µl</td><td class="col1">PstI</td>
 +
</tr>
 +
<tr class="row4">
 +
<td class="col0">36µl dH2O</td><td class="col1"></td>
 +
</tr>
 +
</table></div>
 +
<!-- EDIT13 TABLE [2550-2632] --><ul>
 +
<li class="level1"><div class="li"> incubation at 37°C, 1h</div>
 +
</li>
 +
<li class="level1"><div class="li"> analysis on 1% agarose gel:</div>
 +
</li>
 +
</ul>
 +
<p>
 +
-small band visible for pOP
 +
-nothing for K592009
 +
</p>
 +
<p>
 +
<strong>Inoculation: 3x 5ml LB-Cml wit K592009 (LS)</strong>
 +
</p>
 +
<ul>
 +
<li class="level1"><div class="li"> incubation at 37°C, o/n</div>
 +
</li>
 +
</ul>
 +
</div>
 +
<!-- EDIT11 SECTION "2015/09/05" [2068-2821] -->
 +
<h3 class="sectionedit14">2015/09/06</h3>
 +
<div class="level3">
 +
<p>
 +
<strong> Mini-Prep of K592009 (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 sectionedit15"><table class="inline">
 +
<tr class="row0">
 +
<td class="col0">1</td><td class="col1">77.0 ng/µl</td>
 +
</tr>
 +
<tr class="row1">
 +
<td class="col0">2</td><td class="col1">94.4 ng/µl</td>
 +
</tr>
 +
<tr class="row2">
 +
<td class="col0">3</td><td class="col1">64.6 ng/µl</td>
 +
</tr>
 +
</table></div>
 +
<!-- EDIT15 TABLE [2916-2963] -->
 +
<p>
 +
<strong> Digest of all three preps with EcoRI (JN) </strong>
 +
</p>
 +
<div class="table sectionedit16"><table class="inline">
 +
<tr class="row0">
 +
<th class="col0">amount</th><th class="col1">ingredient</th>
 +
</tr>
 +
<tr class="row1">
 +
<td class="col0">5µl</td><td class="col1">K592009</td>
 +
</tr>
 +
<tr class="row2">
 +
<td class="col0">1µl</td><td class="col1">EcoRI</td>
 +
</tr>
 +
<tr class="row3">
 +
<td class="col0">5µl</td><td class="col1">Cut Smart</td>
 +
</tr>
 +
<tr class="row4">
 +
<td class="col0">39µl</td><td class="col1">dH20</td>
 +
</tr>
 +
</table></div>
 +
<!-- EDIT16 TABLE [3014-3091] --><ul>
 +
<li class="level1"><div class="li"> incubation at 37°C for 1h</div>
 +
</li>
 +
</ul>
 +
</div>
 +
<!-- EDIT14 SECTION "2015/09/06" [2822-] -->
 +
 +
</html>
 +
<!-- Labjournal content ends here -->
 +
{{Freiburg/wiki_content_end}}

Revision as of 10:38, 6 September 2015

""

2015/09/01

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

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

Gel-ex of pOP (JN)

Ligation of pOP+BBa_I13504 (JN)

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

  • 10µl 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

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