Difference between revisions of "Team:NYU Shanghai/Protocols"
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− | + | <b>General Overview:</b> | |
+ | <ol> | ||
+ | <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> | ||
+ | <b>Materials:</b> | ||
+ | <ul> | ||
+ | <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> | ||
+ | </ul> | ||
+ | <b>Lysis Buffer:</b> | ||
+ | <p>For E. coli cell lysis, use a freshly prepared lysozyme solution (10 mg/ml) in 10 mM Tris-HCl, pH 8.0.</p> | ||
+ | <b>Reagent Solution:</b> | ||
+ | <p>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> | ||
+ | <b>Preparing 1mM D-Luciferin:</b> | ||
+ | <p>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 4 uL 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> | ||
+ | <b>Procedure:</b> | ||
+ | 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 25 uL - 30 uL 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> | ||
+ | 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 250-350 uL of reagent solution to each sample of lysis product.</li> | ||
+ | <li>Light should be emitted within two-three seconds.</li> | ||
+ | </ol> | ||
+ | <b>Example Calculations:</b><br> | ||
+ | Lysis Buffer (Desired Total Volume: 15mL)<br> | ||
</div> | </div> | ||
</div> | </div> |
Revision as of 05:15, 16 September 2015