Difference between revisions of "Team:NYU Shanghai/Protocols"
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<p style="font-size: 17px">We built our constructs from pre-made biobrick parts. Our overall conclusion is that 3A assembly is generally inefficient, and an insufficient method for adding small parts (such as a terminator) to a larger construction within pSB1C3. We learned that ratios were extremely important in the process of 3A Assembly, and we made a summary sheet of the equations we used in pre-digest and pre-ligation that accounts for digest dilution and amount needed to ensure results are seen on a gel, not just ligation ratios. We wished we used gibson assembly. </p> | <p style="font-size: 17px">We built our constructs from pre-made biobrick parts. Our overall conclusion is that 3A assembly is generally inefficient, and an insufficient method for adding small parts (such as a terminator) to a larger construction within pSB1C3. We learned that ratios were extremely important in the process of 3A Assembly, and we made a summary sheet of the equations we used in pre-digest and pre-ligation that accounts for digest dilution and amount needed to ensure results are seen on a gel, not just ligation ratios. We wished we used gibson assembly. </p> | ||
<br> | <br> | ||
+ | </div> | ||
+ | <div id="makingColor"> | ||
+ | <h4>Making Color</h4> | ||
+ | |||
+ | <div id="luc" class="collapsed"> | ||
+ | <h5 style="display:inline-block" onclick="expandluc()"><span class="noselect">Luciferase</span></h5> | ||
+ | <div id="lucText" style="display:none"> | ||
+ | <br> | ||
+ | <p><img width="800" src="https://static.igem.org/mediawiki/2015/1/12/NYU_Shanghai_Luciferase_Protein.png"> | ||
+ | <br> | ||
+ | Overview | ||
+ | <ol> | ||
+ | <li>Luciferin substrate must be added.</li> | ||
+ | <li>D-Luciferin is too large of a chemical to cross the plasma membrane of E. Coli so cell lysis is required to extract luciferase.</li> | ||
+ | <li>After cell lysis, the reagent solution can be added to the lysis buffer. Light should be emitted within 5 to 10 seconds of adding the reagent solution.</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> | ||
+ | </ol> | ||
+ | </p> | ||
+ | <p> | ||
+ | <br>Materials | ||
+ | <li>D-Luciferin free acid</li> | ||
+ | <li>ATP</li> | ||
+ | <li>MgSO4 · 7H2O</li> | ||
+ | <li>1M HEPES Buffer</li> | ||
+ | <li>Lysozyme</li> | ||
+ | <li>10 mM Tris-HCl</li> | ||
+ | </p> | ||
+ | <p> | ||
+ | <br>Lysis Buffer | ||
+ | <br>For E. coli cell lysis, use a freshly prepared lysozyme solution (10 mg/ml) in 10 mM Tris-HCl, pH 8.0. | ||
+ | </p> | ||
+ | <p> | ||
+ | <br>Reagent Solution | ||
+ | <br>Prepare using ATP free water. Combine 1 mM luciferin or luciferin salt, 3 mM ATP and 15 mM MgSO4 in 30mM HEPES buffer, pH 7.8. Store substrate solution at -20ºC in polypropylene or glass. | ||
+ | </p> | ||
+ | <p> | ||
+ | <br>Preparing 1mM D-Luciferin | ||
+ | <br>Directions for a 5 mg sample: Dissolve 0.034 gr dithiothreitol in 22 mL of QH2O. Add 0.2 mL of this DTT solution to the 5 mg d-luciferin. Add 4μl of 10M NaOH to dissolve the luciferin. Dilute this into the remaining 21.8 mL of DTT solution and store as aliquots at - 80 ºC in darkness until use. | ||
+ | </p> | ||
+ | <p> | ||
+ | <br>Procedure<br> | ||
+ | Bacterial lysis: | ||
+ | <ol> | ||
+ | <li>After 12-18 hours of inoculation of bacteria expressing luciferase plasmid, pipette 2 mL of cell culture into a clean 2 mL tube. Centrifuge at 10,000 rpm for 1 minute. Pour out liquid into a collection beaker. Continue this process until all of the cell culture (in the inoculation tube) is gone.</li> | ||
+ | <li>Resuspend the pellets in 350 ml of STET buffer (10 mM Tris-HCl with 1 mM EDTA)</li> | ||
+ | <li>Add 25μl - 30μl of lysozyme buffer to the resuspended pellet.</li> | ||
+ | <li>Mix by vortexing for 3 seconds.</li> | ||
+ | <li>Incubate for 2 hours at room temperature.</li> | ||
+ | <li>If the reagent is not added immediately, store the lysed bacteria in the -20 ºC freezer until use.</li> | ||
+ | </ol> | ||
+ | </p> | ||
+ | <p> | ||
+ | Addition of Reagent Solution: | ||
+ | <ol> | ||
+ | <li>Following the above instructions, prepare a 1mM sample of D-Luciferin.</li> | ||
+ | <li>Following the above recipe, prepare the reagent solution.</li> | ||
+ | <li>In a dark room, add about 250-350μl of reagent solution to each sample of lysis product.</li> | ||
+ | <li>Light should be emitted within two-three seconds.</li> | ||
+ | </ol> | ||
+ | </p> | ||
+ | <p> | ||
+ | <br>Example Calculations<br> | ||
+ | Lysis Buffer (Desired Total Volume: 15mL)<br> | ||
+ | <table border="1"> | ||
+ | <tr> | ||
+ | <td><font color="#d66">Chemical Name</font></td> | ||
+ | <td>Tris-HCl</td> | ||
+ | <td>EDTA</td> | ||
+ | <td>NaCl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><font color="#d66">Molecular Weight</font></td> | ||
+ | <td>N/A</td> | ||
+ | <td>292.23 g/mol</td> | ||
+ | <td>58.44 g/mol</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><font color="#d66">Molarity Desired</font></td> | ||
+ | <td>10 mM</td> | ||
+ | <td>1mM</td> | ||
+ | <td>0.1M</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><font color="#d66">Calculation</font></td> | ||
+ | <td>Dilute 1M Tris-HCl:</td> | ||
+ | <td> </td> | ||
+ | <td> </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><font color="#d66">Final Amount</font></td> | ||
+ | <td>150μl (+14.85 mL ddH2O)</td> | ||
+ | <td>0.00438 g</td> | ||
+ | <td>0.08766 g</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </p> | ||
+ | <p> | ||
+ | Lysozyme Solution (Desired Total Volume: 15mL)<br> | ||
+ | <table border="1"> | ||
+ | <tr> | ||
+ | <td><font color="#d66">Lysozyme Solubility</font> </td> | ||
+ | <td>10 mg lysozyme in 1 mL of 10 mM Tris-HCl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><font color="#d66">Desired Amount of Lysozyme Solution to Make</font></td> | ||
+ | <td>15 mL</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><font color="#d66">Amount of Lysozyme Needed</font></td> | ||
+ | <td>10 mg x 15 = <b>150 mg</b></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><font color="#d66">Amount of 10 mM Tris-HCl Needed</font> </td> | ||
+ | <td><b>15 mL</b></td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </p> | ||
+ | <p> | ||
+ | Reagent Solution (Desired total volume: 22 mL)<br> | ||
+ | <table border="1"> | ||
+ | <tr> | ||
+ | <td><font color="#d66">Chemical Name</font></td> | ||
+ | <td>ATP disodium salt trihydrate</td> | ||
+ | <td>MgSO4•7H2O</td> | ||
+ | <td>HEPES Buffer</td> | ||
+ | <td>D-Luciferin free acid</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><font color="#d66">Molecular Weight</font></td> | ||
+ | <td>605.24 g/mol</td> | ||
+ | <td>246.5 g/mol</td> | ||
+ | <td>238.3 g/mol</td> | ||
+ | <td>280.33 g/mol</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><font color="#d66">Molarity Desired</font></td> | ||
+ | <td>3 mM</td> | ||
+ | <td>15 mM</td> | ||
+ | <td>30 mM</td> | ||
+ | <td>1 mM</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><font color="#d66">Calculation</font></td> | ||
+ | <td> </td> | ||
+ | <td> </td> | ||
+ | <td> </td> | ||
+ | <td>Use the 1 mM stock solution created earlier</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><font color="#d66">Final Amount</font></td> | ||
+ | <td>0.039945 g</td> | ||
+ | <td>0.081345 g</td> | ||
+ | <td>0.1573 g</td> | ||
+ | <td>22 mL</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </p> | ||
+ | </p> | ||
+ | </div> | ||
+ | </div> | ||
+ | |||
+ | <div id="chromo" class="collapsed"> | ||
+ | <h5 style="display:inline-block" onclick="expandchromo()"><span class="noselect">Chromoproteins</span></h5> | ||
+ | <br> | ||
+ | <div id="chromoText" style="display:none"> | ||
+ | <p> | ||
+ | <img src="https://static.igem.org/mediawiki/2015/d/df/NYU_Shanghai_Chromo_Procedure.png" width="800"> | ||
+ | </p> | ||
+ | </div> | ||
+ | </div> | ||
</div> | </div> | ||
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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>. | ||
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</div> | </div> | ||
</div> | </div> |
Revision as of 16:34, 17 September 2015