Difference between revisions of "Team:NYU Shanghai/Protocols"
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<li>The luciferase/luciferin reaction at 22.5 ºC theoretically offers the greatest light intensity.</li> | <li>The luciferase/luciferin reaction at 22.5 ºC theoretically offers the greatest light intensity.</li> | ||
<li>Solutions of D-Luciferin should be aliquotted and stored in darkness at -80 ºC</li> | <li>Solutions of D-Luciferin should be aliquotted and stored in darkness at -80 ºC</li> | ||
+ | <li>We were only able to see the color in a very dark room.</li> | ||
</ol> | </ol> | ||
</p> | </p> | ||
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</tr> | </tr> | ||
</table> | </table> | ||
+ | </p> | ||
+ | <p> | ||
+ | Controls | ||
+ | <li>No arabinose added during inoculation</li> | ||
+ | <li>Use bacteria without luciferase plasmid and go through steps to induce color</li> | ||
</p> | </p> | ||
</p> | </p> | ||
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<div id="chromoText" style="display:none"> | <div id="chromoText" style="display:none"> | ||
<p> | <p> | ||
− | <img src="https://static.igem.org/mediawiki/2015/d/df/NYU_Shanghai_Chromo_Procedure.png" width="800"> | + | <h6><font color="#d66">Building our Construct: from biobrick parts in the kit</font></h6> |
+ | <p>Note: If using construct with pBAD promoter, DO NOT USE SOC MEDIA. Glucose inhibits the uptake of arabinose, and will inhibit promoter induction. | ||
+ | <br>Note: We should have used PCR to amplify linearized backbone. | ||
+ | <br>Note: Always use gel electrophoresis to check digest results.</p> | ||
+ | <br><img src="https://static.igem.org/mediawiki/2015/d/df/NYU_Shanghai_Chromo_Procedure.png" width="800"> | ||
+ | <br><br><br> | ||
+ | <h6><font color="#d66">Building our Construct: from IDT gBlocks</font></h6> | ||
+ | <p>Note: We recommend adding a reporter gene to the construct.</p> | ||
+ | <br><img src="https://static.igem.org/mediawiki/2015/7/71/NYU_Shanghai_IDTprocedure.png" width="550"> | ||
+ | <br><br><br> | ||
+ | <h6><font color="#d66">Expressing XJTLU's Construct</font></h6> | ||
+ | <br><img src="https://static.igem.org/mediawiki/2015/thumb/9/9a/NYU_Shanghai_Chromo_Procedure_2.png/573px-NYU_Shanghai_Chromo_Procedure_2.png"> | ||
</p> | </p> | ||
</div> | </div> | ||
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</table> | </table> | ||
</p> | </p> | ||
+ | <p>Controls | ||
+ | <li>DNA with known sites for the enzyme</li> | ||
+ | <li>If control DNA cleaved and experimental DNA resists cleavage, the two DNAs can be mixed to determine if an inhibitor is present in the experimental sample</li> | ||
+ | </p> | ||
</div> | </div> | ||
</div> | </div> | ||
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<li>Visualize the gel and record the results.</li> | <li>Visualize the gel and record the results.</li> | ||
</ol> | </ol> | ||
+ | |||
+ | <br><p>Controls | ||
+ | <li>Uncut plasmid</li> | ||
+ | <li>Uncut insert DNA</li> | ||
+ | <li>Ladder DNA</li> | ||
+ | </p> | ||
</div> | </div> | ||
</div> | </div> | ||
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<td>Nuclease-free water</td> | <td>Nuclease-free water</td> | ||
<td>10μl or none</td> | <td>10μl or none</td> | ||
+ | <td></td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <br> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <td><font color="#d66">Initial Denaturation</font></td> | ||
+ | <td>98C</td> | ||
+ | <td>30s</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><font color="#d66">25 cycles</font></td> | ||
+ | <td>98C</td> | ||
+ | <td>15s</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Annealing temp 1</td> | ||
+ | <td>59.5C</td> | ||
+ | <td>30s</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Annealing temp 2</td> | ||
+ | <td>56.3C</td> | ||
+ | <td>30s</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Annealing temp 3</td> | ||
+ | <td>53.7C</td> | ||
+ | <td>30s</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Extension</td> | ||
+ | <td>72C</td> | ||
+ | <td>60s</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><font color="#d66">Final Extension</font></td> | ||
+ | <td>72C</td> | ||
+ | <td>2m</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><font color="#d66">Hold</font></td> | ||
+ | <td>4C</td> | ||
<td></td> | <td></td> | ||
</tr> | </tr> | ||
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<div id="PCRcleanText" style="display:none"> | <div id="PCRcleanText" style="display:none"> | ||
<p>We used <a href="https://static.igem.org/mediawiki/2015/d/d1/NYU_Shanghai_Tianquick.pdf">TIANquick Mini Purification Kit</a>. | <p>We used <a href="https://static.igem.org/mediawiki/2015/d/d1/NYU_Shanghai_Tianquick.pdf">TIANquick Mini Purification Kit</a>. | ||
+ | <ol> | ||
+ | <li>Add ethanol (96-100%) to Buffer PW before use (see bottle label | ||
+ | for volume).</li> | ||
+ | <li>Column equilibration: add 500μl Buffer BL to the Spin Column CB1 (put Spin Column CB1 into a collection tube). Centrifuge for 1min at 12,000 rpm. Discard the flow-through, and then place Spin Column CB1 back into the collection tube.</li> | ||
+ | <li>Add 5 volumes of Buffer PB to 1 volume of the PCR reaction or enzymatic reaction and mix.</li> | ||
+ | <li>Transfer the mixture to the Spin Column CB1, incubate at room temperature for 2min. Centrifuge for 30-60s at 12,000rpm. Discard the flow-through, and then place Spin Column CB1 back into the same collection tube. | ||
+ | <br>The maximum loading volume of the column is 800μl. For sample volumes greater than 800 μl simply load again.</li> | ||
+ | <li>Add 600 μl Buffer PW (ensure that ethanol has been added) to the Spin Column CB1 and centrifuge for 30-60s at 12,000 rpm. Discard the flow-through, and place Spin Column CB1 back in the same collection tube. | ||
+ | <br>Note: If the purified DNA is used for the subsequent salt sensitive experiments, such as ligation or sequencing experiment, it is suggested to stand for 2-5min after adding Buffer PW, and then centrifuge.</li> | ||
+ | <li>Repeat step 4.</li> | ||
+ | <li>Centrifuge at 12,000 rpm for 2min to remove residual Buffer PW. Discard the flow-through, and allow the column to air dry with the cap open for several minutes to dry the membrane.</li> | ||
+ | <li>Place the Spin Column CB1 in a clean 1.5ml microcentrifuge tube. Add 20μl Buffer EB to the center of membrane, incubate for 2min, and centrifuge for 2min at 12,000 rpm</li> | ||
+ | </ol> | ||
+ | </p> | ||
</div> | </div> | ||
</div> | </div> |
Latest revision as of 16:56, 18 September 2015