Difference between revisions of "Team:NYU Shanghai/Protocols"
Line 452: | Line 452: | ||
<div id="lucText" style="display:none"> | <div id="lucText" style="display:none"> | ||
<p> | <p> | ||
− | + | Overview | |
<ol> | <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>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> | ||
Line 459: | Line 459: | ||
<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> | ||
</ol> | </ol> | ||
− | < | + | <br>Materials |
<ul> | <ul> | ||
<li>D-Luciferin free acid</li> | <li>D-Luciferin free acid</li> | ||
Line 470: | Line 470: | ||
<b>Lysis Buffer:</b> | <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> | <p>For E. coli cell lysis, use a freshly prepared lysozyme solution (10 mg/ml) in 10 mM Tris-HCl, pH 8.0.</p> | ||
− | < | + | <br>Reagent Solution |
<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> | <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> | ||
− | < | + | <br>Preparing 1mM D-Luciferin |
<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> | <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> | ||
− | < | + | <br>Procedure<br> |
Bacterial lysis: | Bacterial lysis: | ||
<ol> | <ol> | ||
Line 491: | Line 491: | ||
<li>Light should be emitted within two-three seconds.</li> | <li>Light should be emitted within two-three seconds.</li> | ||
</ol> | </ol> | ||
− | < | + | <br>Example Calculations<br> |
Lysis Buffer (Desired Total Volume: 15mL)<br> | Lysis Buffer (Desired Total Volume: 15mL)<br> | ||
<table border="1"> | <table border="1"> |
Revision as of 17:00, 16 September 2015