Difference between revisions of "Team:NYU Shanghai/Protocols"
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<br><br>Materials | <br><br>Materials | ||
<li>2µl resuspended DNA</li> | <li>2µl resuspended DNA</li> | ||
− | <li>Competent cells ( | + | <li>Competent cells (50μl per transformation)</li> |
<li>Ice</li> | <li>Ice</li> | ||
<li>2ml tube</li> | <li>2ml tube</li> | ||
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<tr> | <tr> | ||
<td>10X NEB Buffer</td> | <td>10X NEB Buffer</td> | ||
− | <td> | + | <td>5μl (1X)</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
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<table border="1"> | <table border="1"> | ||
<tr style="font-weight: bold;"> | <tr style="font-weight: bold;"> | ||
− | <td> | + | <td><font color="#d66">Components</font></td> |
− | <td> | + | <td><font color="#d66">20μl REACTION</font></td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>10X T4 DNA Ligase Buffer*</td> | <td>10X T4 DNA Ligase Buffer*</td> | ||
− | <td> | + | <td>2μl</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>Vector DNA (4 kb)</td> | <td>Vector DNA (4 kb)</td> | ||
− | <td> | + | <td>50ng (0.020 pmol)</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>Insert DNA (1 kb)</td> | <td>Insert DNA (1 kb)</td> | ||
− | <td>37. | + | <td>37.5ng (0.060 pmol)</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>Nuclease-free water</td> | <td>Nuclease-free water</td> | ||
− | <td>to | + | <td>to 20μl</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>T4 DNA Ligase</td> | <td>T4 DNA Ligase</td> | ||
− | <td> | + | <td>1μl</td> |
</tr> | </tr> | ||
</table> | </table> | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
<li>Ligation temperature and times vary</li> | <li>Ligation temperature and times vary</li> | ||
<table border="1" width="600"> | <table border="1" width="600"> | ||
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<tr> | <tr> | ||
<td>For 3A Assembly</td> | <td>For 3A Assembly</td> | ||
− | <td> </td> | + | <td>Room temperature for an hour, then overnight in 4degree</td> |
</tr> | </tr> | ||
</table> | </table> | ||
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<li>Chill on ice and transform 1-5 μl of the reaction into 50 μl competent cells.</li> | <li>Chill on ice and transform 1-5 μl of the reaction into 50 μl competent cells.</li> | ||
</ol> | </ol> | ||
+ | |||
+ | <br><p>Controls | ||
+ | <li>Reaction mixture with no insert DNA</li> | ||
+ | <li>Reaction mixture with no insert DNA and no ligase</li> | ||
+ | </p>> | ||
</div> | </div> | ||
</div> | </div> | ||
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<tr> | <tr> | ||
<td><font color="#d66">Component</font></td> | <td><font color="#d66">Component</font></td> | ||
− | <td><font color="#d66"> | + | <td><font color="#d66">50μl Reaction</font></td> |
<td><font color="#d66">Concentration</font></td> | <td><font color="#d66">Concentration</font></td> | ||
</tr> | </tr> | ||
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<tr> | <tr> | ||
<td>Nuclease-free water</td> | <td>Nuclease-free water</td> | ||
− | <td> | + | <td>10μl or none</td> |
<td></td> | <td></td> | ||
</tr> | </tr> | ||
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<p> | <p> | ||
<br>Preparing 1mM D-Luciferin | <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 | + | <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> | ||
<p> | <p> | ||
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<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>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>Resuspend the pellets in 350 ml of STET buffer (10 mM Tris-HCl with 1 mM EDTA)</li> | ||
− | <li>Add | + | <li>Add 25μl - 30μl of lysozyme buffer to the resuspended pellet.</li> |
<li>Mix by vortexing for 3 seconds.</li> | <li>Mix by vortexing for 3 seconds.</li> | ||
<li>Incubate for 2 hours at room temperature.</li> | <li>Incubate for 2 hours at room temperature.</li> | ||
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<li>Following the above instructions, prepare a 1mM sample of D-Luciferin.</li> | <li>Following the above instructions, prepare a 1mM sample of D-Luciferin.</li> | ||
<li>Following the above recipe, prepare the reagent solution.</li> | <li>Following the above recipe, prepare the reagent solution.</li> | ||
− | <li>In a dark room, add about 250- | + | <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> | <li>Light should be emitted within two-three seconds.</li> | ||
</ol> | </ol> | ||
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<tr> | <tr> | ||
<td><font color="#d66">Final Amount</font></td> | <td><font color="#d66">Final Amount</font></td> | ||
− | <td> | + | <td>150μl (+14.85 mL ddH2O)</td> |
<td>0.00438 g</td> | <td>0.00438 g</td> | ||
<td>0.08766 g</td> | <td>0.08766 g</td> |
Revision as of 14:51, 17 September 2015