Team:Marburg:Project:Notebook:April
From 2014.igem.org
(Difference between revisions)
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</div> | </div> | ||
</fieldset> | </fieldset> | ||
+ | |||
+ | <!-- ROMAN --> | ||
+ | |||
+ | <fieldset class="exp14"> | ||
+ | <legend><a name="exp14.1">14.1 PCR of PheA-Arc1p-C-8x</a></legend> | ||
+ | <div class="aim"> | ||
+ | <p>Aim: Amplify PheA-Arc1p-C-8x fragment for Gibson Assembly in pET-28a</p> | ||
+ | </div> | ||
+ | <div class="exp-content"> | ||
+ | <p>The PCR with TG_PheA-Arc1p-C-8x FP und TG_PheA-Arc1p-C-8x RP should generate the 2346 bp fragment for Gibson Assembly from the synthesized template.</p> | ||
+ | <p>The used Primer concentration was 1 | ||
+ | µM, the concentration of the template was 1 ng/µL and we used a hot start to avoid unspecific annealing of primers.</p> | ||
+ | <table width="100%" border="1"> | ||
+ | <tr> | ||
+ | <th scope="col">Content</th> | ||
+ | <th scope="col">Volume [µL]</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th scope="row">Water</th> | ||
+ | <td>29.5</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th scope="row">5x GC Buffer</th> | ||
+ | <td>10</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th scope="row">DMSO</th> | ||
+ | <td>2.5</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th scope="row">TG_PheA-Arc1p-C-8x FP</th> | ||
+ | <td>2.5</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th scope="row">TG_PheA-Arc1p-C-8x RP</th> | ||
+ | <td>2.5</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th scope="row">synthesized template</th> | ||
+ | <td>1</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th scope="row">dNTPs</th> | ||
+ | <td>1</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th scope="row">Phusion-Polymerase</th> | ||
+ | <td>1</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th scope="row">Total Volume</th> | ||
+ | <td>50</td> | ||
+ | </tr> | ||
+ | </table><br /> | ||
+ | <table width="100%" border="1"> | ||
+ | <tr> | ||
+ | <th scope="col">Step</th> | ||
+ | <th scope="col">Temperature [°C]</th> | ||
+ | <th scope="col">Time [min:sec]</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th scope="row">1</th> | ||
+ | <td>98</td> | ||
+ | <td>2:00</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th scope="row">2</th> | ||
+ | <td>add Polymerase</td> | ||
+ | <td> </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th scope="row">3</th> | ||
+ | <td>98</td> | ||
+ | <td>0:10</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th scope="row">4</th> | ||
+ | <td>67.5</td> | ||
+ | <td>0:30</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th scope="row">5</th> | ||
+ | <td>72</td> | ||
+ | <td>1:40</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th scope="row">6</th> | ||
+ | <td>go to 3</td> | ||
+ | <td>32x</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th scope="row">7</th> | ||
+ | <td>72</td> | ||
+ | <td>10:00</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th scope="row">8</th> | ||
+ | <td>4</td> | ||
+ | <td>hold</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>The amplified fragment was purified on an agarose gel on which it showed the correct size. It was extracted from the gel for further use in a Gibson Assembly yielding a concentration of 258 ng/µL.</p> | ||
+ | </div> | ||
+ | </fieldset> | ||
+ | |||
+ | <fieldset class="exp14"> | ||
+ | <legend><a name="exp14.2">14.2 Digestion of pET-28a</a></legend> | ||
+ | <div class="aim"> | ||
+ | <p>Aim: Linearize pET-28a to prepare it for Gibson Assembly</p> | ||
+ | </div> | ||
+ | <div class="exp-content"> | ||
+ | <p>The pET-28a vector was cut with XhoI and NdeI in order to create the ends needed for the integration of the prepared PheA-Arc1p-C-8x construct via Gibson Assembly</p> | ||
+ | <table width="100%" border="1"> | ||
+ | <tr> | ||
+ | <th scope="col">Content</th> | ||
+ | <th scope="col">Volume [µL]</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th scope="row">Water</th> | ||
+ | <td>34.2</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th scope="row">CutSmart Buffer</th> | ||
+ | <td>5</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th scope="row">pET-28a (128 ng/µL)</th> | ||
+ | <td>7.81</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th scope="row">NdeI</th> | ||
+ | <td>1.5</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th scope="row">XhoI</th> | ||
+ | <td>1.5</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th scope="row">Total Volume</th> | ||
+ | <td>50</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>The reaction mixture was incubated for 3 h at 37 °C and 350 rpm. The resulting linearized vector was purified using an agarose gel from which it was extracted for further use yielding a concentration of 24 ng/µL.</p> | ||
+ | </div> | ||
+ | </fieldset> | ||
+ | |||
+ | <fieldset class="exp14"> | ||
+ | <legend><a name="exp14.3">14.3 Gibson Assembly</a></legend> | ||
+ | <div class="aim"> | ||
+ | <p>Aim: Integrate PheA-Arc1p-C-8x into linearized pET-28a(XhoI,NdeI) via Gibson Assembly</p> | ||
+ | </div> | ||
+ | <div class="exp-content"> | ||
+ | <table width="100%" border="1"> | ||
+ | <tr> | ||
+ | <th scope="col">Content</th> | ||
+ | <th scope="col">Volume [µL]</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th scope="row">Water</th> | ||
+ | <td>1.46</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th scope="row">pET-28a(XhoI,NdeI) (24 ng/µL)</th> | ||
+ | <td>4.17</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th scope="row">PheA-Arc1p-C-8x (30 ng/µL)</th> | ||
+ | <td>4.37</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th scope="row">2x Gibson Mastermix</th> | ||
+ | <td>10</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th scope="row">Total Volume</th> | ||
+ | <td>20</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>The reaction mixture was incubated for 1 h at 50 °C and 350 rpm. E. coli Top10 electrocompetent cells were transformed with a 1:3 and a 1:6 dilution of the mixture, incubated for 1 h at 37 °C and plated out on LB-Kan<sup>50</sup> plates that were incubated overnight at 37 °C.</p> | ||
+ | </div> | ||
+ | </fieldset> | ||
+ | |||
+ | <!-- //ROMAN --> | ||
+ | |||
+ | |||
<fieldset class="exp14"> | <fieldset class="exp14"> | ||
<legend><a name="exp14.2">14.2 Miniprep of <i>E.coli</i> pMA12</a></legend> | <legend><a name="exp14.2">14.2 Miniprep of <i>E.coli</i> pMA12</a></legend> | ||
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</div> | </div> | ||
</fieldset> | </fieldset> | ||
+ | |||
+ | <!-- ROMAN --> | ||
+ | <fieldset class="exp14"> | ||
+ | <legend><a name="exp14.4">14.4 Overnight culture</a></legend> | ||
+ | <div class="aim"> | ||
+ | <p>Aim: Amplify Plasmid in an overnight culture</p> | ||
+ | </div> | ||
+ | <div class="exp-content"> | ||
+ | <p>Six clones of the transformed Top10 cells were picked and used to inoculate overnight cultures (6 x 5 mL).</p> | ||
+ | </div> | ||
+ | </fieldset> | ||
+ | <!-- ROMAN --> | ||
+ | |||
</div> | </div> | ||
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</div> | </div> | ||
</fieldset> | </fieldset> | ||
+ | |||
+ | <fieldset class="exp14"> | ||
+ | <legend><a name="exp14.5">14.5 Preparation of plasmids</a></legend> | ||
+ | <div class="aim"> | ||
+ | <p>Aim: Purify the amplified plasmids from overnight cultures</p> | ||
+ | </div> | ||
+ | <div class="exp-content"> | ||
+ | <p>The plasmids were purified from the overnight cultures using Sigma GenElute Plasmid Miniprep Kit following the included protocol. The plasmids were eluted using 45 µL water.</p> | ||
+ | </div> | ||
+ | </fieldset> | ||
+ | |||
+ | <fieldset class="exp14"> | ||
+ | <legend><a name="exp14.6">14.6 Transformation of BL21 cells</a></legend> | ||
+ | <div class="aim"> | ||
+ | <p>Aim: Transform E. coli BL21(DE3) with PheA-Arc1p-C-8x-pET28a</p> | ||
+ | </div> | ||
+ | <div class="exp-content"> | ||
+ | <p>40 µL E. coli BL21(DE3) electrocompetent cells were transformed with 1 ng of the previously purified plasmid PheA-Arc1p-C-8x-pET28a from two different clones, incubated for 1 h at 37 °C and plated out on LB-Kan<sup>50</sup> plates that were incubated overnight at 37 °C.</p> | ||
+ | </div> | ||
+ | </fieldset> | ||
+ | |||
</div> | </div> | ||
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</div> | </div> | ||
</fieldset> | </fieldset> | ||
+ | |||
+ | <fieldset class="exp14"> | ||
+ | <legend><a name="exp14.7">14.7 Overnight culture</a></legend> | ||
+ | <div class="aim"> | ||
+ | <p>Aim: Inoculate Overnight culture of PheA-Arc1p-C-8x-pET28a</p> | ||
+ | </div> | ||
+ | <div class="exp-content"> | ||
+ | <p>5 mL of LB-Kan<sup>50</sup> medium were inoculated with a clone from 14.6 bearing the PheA-Arc1p-C-8x-pET28a plasmid.</p> | ||
+ | </div> | ||
+ | </fieldset> | ||
+ | |||
</div> | </div> | ||
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</div> | </div> | ||
</fieldset> | </fieldset> | ||
+ | |||
+ | <!-- ROMAN --> | ||
+ | |||
+ | <fieldset class="exp14"> | ||
+ | <legend><a name="exp14.8">14.8 Test Expression</a></legend> | ||
+ | <div class="aim"> | ||
+ | <p>Aim: Test the expression of PheA-Arc1p-C-8x</p> | ||
+ | </div> | ||
+ | <div class="exp-content"> | ||
+ | <p>Using the preculture from 14.7 60 mL of LB-Kan<sup>50</sup> medium were inoculated with 600 µL of the preculture and incubated at 37 °C and 220 rpm until OD<sub>600</sub> reached 0.5. The culture was cooled down to 18 °C and the protein production was induced with 12 µL of 500 mM IPTG. Over night the culture was incubated at 18 °C and 220 rpm. A glycerolstock of the strain containing the PheA-Arc1p-C-8x-pET28a plasmid was stored at -80 °C.</p> | ||
+ | </div> | ||
+ | </fieldset> | ||
+ | |||
+ | <!-- ROMAN --> | ||
+ | |||
</div> | </div> | ||
Line 1,760: | Line 2,005: | ||
</div> | </div> | ||
</fieldset> | </fieldset> | ||
+ | |||
+ | <!-- ROMAN --> | ||
+ | <fieldset class="exp14"> | ||
+ | <legend><a name="exp14.9">14.9 Purification of test expression</a></legend> | ||
+ | <div class="aim"> | ||
+ | <p>Aim: Purify PheA-Arc1p-C-8x on a small scale</p> | ||
+ | </div> | ||
+ | <div class="exp-content"> | ||
+ | <p>The cell culture from 14.8 was centrifuged (17000 rpm, 20 min, 4 °C) and the pellet resuspended in 3 mL buffer A. Lysozyme was added to a final concentration of 1 mg/mL and incubated on ice for 30 min. Afterwards the cells were lysed using ultra sound (2x 18 pulses, 30 s on ice inbetween). To clear the suspension of cell debris it was centrifuged (13000 rpm, 15 min, 4 °C). The supernatant was purified using QIAGEN Ni-NTA Spin Columns. An SDS-PAGE gel showed that the protein is produced and can be purified using Ni affinity chromatography.</p> | ||
+ | <p>60 mL LB-Kan<sup>50</sup> medium was inoculated from the glycerolstock prepared in 14.8 and incubated at 37 °C and 220 rpm over night.</p> | ||
+ | </div> | ||
+ | </fieldset> | ||
+ | <!-- // ROMAN --> | ||
</div> | </div> | ||
- | |||
+ | <!-- ROMAN --> | ||
<div class="notebooky-entry"> | <div class="notebooky-entry"> | ||
- | <h2 class="title"> | + | <h2 class="title"> |
- | + | <a name="17.04.2014">17.04.2014</a> | |
- | </h2> | + | </h2> |
- | <fieldset class=" | + | <fieldset class="exp14"> |
- | + | <legend><a name="exp14.10">14.10 Expression of PheA-Arc1p-C-8x</a></legend> | |
- | + | <div class="aim"> | |
- | + | <p>Aim: Express PheA-Arc1p-C-8x for further use</p> | |
- | + | </div> | |
- | + | <div class="exp-content"> | |
- | + | <p>10 x 500 mL LB-Kan<sup>50</sup> medium were inoculated with 5 mL of the overnight culture prepared in 14.9 and incubated at 37 °C and 220 rpm until OD<sub>600</sub> reached 0.5. The flasks were cooled to 18 °C and protein production was induced by adding 100 µL 500 mM IPTG. The cultures were incubated over night at 18 °C and 220 rpm. Afterwards the cells were collected by centrifugation (6000 rpm, 20 min, 4 °C), resuspended in buffer A and stored at -20 °C until further use.</p> | |
- | + | </div> | |
- | + | </fieldset> | |
- | + | ||
- | + | ||
- | + | ||
- | + | ||
</div> | </div> | ||
- | </fieldset> | + | <!-- %% ROMAN --> |
+ | |||
+ | |||
+ | <!-- 22.04.14 --> | ||
+ | |||
+ | <div class="notebooky-entry"> | ||
+ | <h2 class="title"> | ||
+ | <a name="22.04.2014">22.04.2014</a> | ||
+ | </h2> | ||
+ | <fieldset class="exp13"> | ||
+ | <legend><a name="exp13.29">13.29 Miniprep of Transformed Yeast <em>S.cerevisiae </em>and Trafo of <em>E.coli</em> DH5Alpha</a></legend> | ||
+ | <div class="Aim"> | ||
+ | <p>Aim: Increase the amount of plasmid DNA to perform restriction and further cloning steps.</p> | ||
+ | </div> | ||
+ | <div class="exp-content"> | ||
+ | <p>Miniprep of Transformed Yeast <em>S.cerevisiae</em> from 21.04.2014 and Trafo of <em>E.coli</em> DH5Alpha.<br /> | ||
+ | Miniprep was performed according to the miniprep protocol (Omega). | ||
+ | </p> | ||
+ | <p>Plasmid pMa12 with 3 inserts (cat, amyE and gfp) with removed NcoI restriction sites.<br /> | ||
+ | Concentration after plasmid prep= 7.5 ng/µL<br /> | ||
+ | Transformation of <i>E.coli</i> DH5α with 5 µL DNA (37.5 ng)<br /> | ||
+ | Over night incubation on LB-Amp plates.</p> | ||
+ | </div> | ||
+ | </fieldset> | ||
+ | <!-- ROMAN --> | ||
+ | <fieldset class="exp14"> | ||
+ | <legend><a name="exp14.11">14.11 Purification of PheA-Arc1p-C-8x</a></legend> | ||
+ | <div class="aim"> | ||
+ | <p>Aim: Purify PheA-Arc1p-C-8x in three steps</p> | ||
+ | </div> | ||
+ | <div class="exp-content"> | ||
+ | <p>The resuspended cells from 14.10 were lysed using a french press. The lysate was incubated on ice with DNAseA and RNAseI for 10 min and centrifuged (27000 rpm, 45 min, 4 °C). The supernatant was filtered (0.45 µm) and PheA-Arc1p-C-8x purified from the cleared solution by NiNTA affinity chromatography. The combined elution fractions containing crude PheA-Arc1p-C were concentrated to a volume of 2 mL and further purified using an anion exchange column to get rid of possible tRNA contaminations. Finally the buffer was changed to dialysis buffer and the protein concentrated and stored at -80 °C in aliquods for further use.</p> | ||
+ | </div> | ||
+ | </fieldset> | ||
+ | <!-- %% ROMAN --> | ||
</div> | </div> | ||
Line 1,887: | Line 2,175: | ||
</div> | </div> | ||
</fieldset> | </fieldset> | ||
+ | |||
+ | <!-- ROMAN --> | ||
+ | <fieldset class="exp14"> | ||
+ | <legend><a name="exp14.12">14.12 Creating PheA-Arc1p-C-4x</a></legend> | ||
+ | <div class="aim"> | ||
+ | <p>Aim: Using Round-the-horn mutagenesis to create PheA-Arc1p-C-4x-pET28a</p> | ||
+ | </div> | ||
+ | <div class="exp-content"> | ||
+ | The PCR with 5' phosphorylated TG_2xGSSG FP und TG_2xGSSG RP should generate the 7545 bp fragment for ligation from the methylated template PheA-Arc1p-C-8x-pET28a from 14.5.</p> | ||
+ | <p>The used Primer concentration was 1 | ||
+ | µM, the concentration of the template was 1 ng/µL and we used a hot start to avoid unspecific annealing of primers.</p> | ||
+ | <table width="100%" border="1"> | ||
+ | <tr> | ||
+ | <th scope="col">Content</th> | ||
+ | <th scope="col">Volume [µL]</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th scope="row">Water</th> | ||
+ | <td>29.5</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th scope="row">5x GC Buffer</th> | ||
+ | <td>10</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th scope="row">DMSO</th> | ||
+ | <td>2.5</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th scope="row">TG_2xGSSG FP</th> | ||
+ | <td>2.5</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th scope="row">TG_2xGSSG RP</th> | ||
+ | <td>2.5</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th scope="row">PheA-Arc1p-C-8x-pET28a</th> | ||
+ | <td>1</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th scope="row">dNTPs</th> | ||
+ | <td>1</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th scope="row">Phusion-Polymerase</th> | ||
+ | <td>1</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th scope="row">Total Volume</th> | ||
+ | <td>50</td> | ||
+ | </tr> | ||
+ | </table><br /> | ||
+ | <table width="100%" border="1"> | ||
+ | <tr> | ||
+ | <th scope="col">Step</th> | ||
+ | <th scope="col">Temperature [°C]</th> | ||
+ | <th scope="col">Time [min:sec]</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th scope="row">1</th> | ||
+ | <td>98</td> | ||
+ | <td>2:00</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th scope="row">2</th> | ||
+ | <td>add Polymerase</td> | ||
+ | <td> </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th scope="row">3</th> | ||
+ | <td>98</td> | ||
+ | <td>0:10</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th scope="row">4</th> | ||
+ | <td>67.5</td> | ||
+ | <td>0:30</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th scope="row">5</th> | ||
+ | <td>72</td> | ||
+ | <td>4:00</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th scope="row">6</th> | ||
+ | <td>go to 3</td> | ||
+ | <td>32x</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th scope="row">7</th> | ||
+ | <td>72</td> | ||
+ | <td>10:00</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th scope="row">8</th> | ||
+ | <td>4</td> | ||
+ | <td>hold</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>The amplified fragment was purified on an agarose gel on which it showed the correct size. The extracted linear template was then digested to degrade the original unmutated plasmid using DpnI.</p> | ||
+ | <table width="100%" border="1"> | ||
+ | <tr> | ||
+ | <th scope="col">Content</th> | ||
+ | <th scope="col">Volume [µL]</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th scope="row">10x CutSmart Buffer</th> | ||
+ | <td>3.6</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th scope="row">Plasmid</th> | ||
+ | <td>30</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th scope="row">DpnI</th> | ||
+ | <td>3</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <th scope="row">Total Volume</th> | ||
+ | <td>36.6</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <p>The reaction mixture was incubated at 37 °C and 300 rpm for 1 h and afterwards heated to 80 °C for 20 min</p> | ||
+ | |||
+ | <p>In order to create the circular plasmid 100 ng of the linear template were mixed with 1 µL 10x T4-Ligasebuffer, 1 µL T4-Ligase and water to give a total volume of 10 µL. This mixture was incubated for 9 h at 16 °C. The resulting plasmid was dialysed with water to decrease the salt concentration for the following transformation of E. coli Top10 electrocompetent cells with the plasmid.</p> | ||
+ | </div> | ||
+ | </fieldset> | ||
+ | <!-- %% ROMAN --> | ||
+ | |||
</div> | </div> | ||
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</div> | </div> | ||
</fieldset> | </fieldset> | ||
+ | <!-- ROMAN --> | ||
+ | |||
+ | <fieldset class="exp14"> | ||
+ | <legend><a name="exp14.13">14.13 Overnight culture</a></legend> | ||
+ | <div class="aim"> | ||
+ | <p>Aim: Amplify Plasmid in an overnight culture</p> | ||
+ | </div> | ||
+ | <div class="exp-content"> | ||
+ | <p>Six clones of the transformed Top10 cells from 14.12 were picked and used to inoculate overnight cultures (6 x 5 mL).</p> | ||
+ | </div> | ||
+ | </fieldset> | ||
+ | |||
+ | <!-- %% ROMAN --> | ||
+ | |||
+ | |||
</div> | </div> | ||
Line 2,138: | Line 2,571: | ||
</div> | </div> | ||
</fieldset> | </fieldset> | ||
+ | |||
+ | <!-- ROMAN --> | ||
+ | |||
+ | <fieldset class="exp14"> | ||
+ | <legend><a name="exp14.14">14.14 Preparation of plasmids</a></legend> | ||
+ | <div class="aim"> | ||
+ | <p>Aim: Purify the amplified plasmids from overnight cultures</p> | ||
+ | </div> | ||
+ | <div class="exp-content"> | ||
+ | <p>The plasmids were purified from the overnight cultures created in 14.13 using Sigma GenElute Plasmid Miniprep Kit following the included protocol. The plasmids were eluted using 45 µL water.</p> | ||
+ | </div> | ||
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+ | <fieldset class="exp14"> | ||
+ | <legend><a name="exp14.15">14.15 Transformation of BL21 cells</a></legend> | ||
+ | <div class="aim"> | ||
+ | <p>Aim: Transform E. coli BL21(DE3) with PheA-Arc1p-C-4x-pET28a</p> | ||
+ | </div> | ||
+ | <div class="exp-content"> | ||
+ | <p>40 µL E. coli BL21(DE3) electrocompetent cells were transformed with 1 ng of the previously purified plasmid PheA-Arc1p-C-4x-pET28a from two different clones, incubated for 1 h at 37 °C and plated out on LB-Kan<sup>50</sup> plates that were incubated overnight at 37 °C.</p> | ||
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+ | <fieldset class="exp14"> | ||
+ | <legend><a name="exp14.16">14.16 Overnight culture</a></legend> | ||
+ | <div class="aim"> | ||
+ | <p>Aim: Inoculate Overnight culture of PheA-Arc1p-C-4x-pET28a</p> | ||
+ | </div> | ||
+ | <div class="exp-content"> | ||
+ | <p>5 mL of LB-Kan<sup>50</sup> medium were inoculated with a clone from 14.15 bearing the PheA-Arc1p-C-4x-pET28a plasmid.</p> | ||
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+ | <fieldset class="exp14"> | ||
+ | <legend><a name="exp14.17">14.17 Test Expression</a></legend> | ||
+ | <div class="aim"> | ||
+ | <p>Aim: Test the expression of PheA-Arc1p-C-4x</p> | ||
+ | </div> | ||
+ | <div class="exp-content"> | ||
+ | <p>Using the preculture from 14.16 60 mL of LB-Kan<sup>50</sup> medium were inoculated with 600 µL of the preculture and incubated at 37 °C and 220 rpm until OD<sub>600</sub> reached 0.5. The culture was cooled down to 18 °C and the protein production was induced with 12 µL of 500 mM IPTG. Over night the culture was incubated at 18 °C and 220 rpm. A glycerolstock of the strain containing the PheA-Arc1p-C-4x-pET28a plasmid was stored at -80 °C.</p> | ||
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Revision as of 18:37, 14 October 2014
Notebook: April