Difference between revisions of "Team:NYU Shanghai/Protocols"
Line 371: | Line 371: | ||
<br> | <br> | ||
<div id="SOCText" style="display:none"> | <div id="SOCText" style="display:none"> | ||
+ | <p> | ||
Materials | Materials | ||
− | |||
<li>0.5% (w/v) yeast extract</li> | <li>0.5% (w/v) yeast extract</li> | ||
<li>2% (w/v) tryptone</li> | <li>2% (w/v) tryptone</li> | ||
Line 378: | Line 378: | ||
<li>2.5 mM KCl</li> | <li>2.5 mM KCl</li> | ||
<li>20 mM MgSO4</li> | <li>20 mM MgSO4</li> | ||
− | + | <br><br>Per liter: | |
<li>5 g yeast extract</li> | <li>5 g yeast extract</li> | ||
<li>20 g tryptone</li> | <li>20 g tryptone</li> | ||
Line 384: | Line 384: | ||
<li>0.186 g KCl</li> | <li>0.186 g KCl</li> | ||
<li>2.4 g MgSO4</li> | <li>2.4 g MgSO4</li> | ||
− | < | + | <br><br> |
+ | </p> | ||
<p><em>Note:</em> Some formulations of SOB use 10 mM MgCl2 and 10 mM MgSO4 instead of 20 mM MgSO4. | <p><em>Note:</em> Some formulations of SOB use 10 mM MgCl2 and 10 mM MgSO4 instead of 20 mM MgSO4. | ||
SOB medium is also available dry premixed from Difco, 0443-17.<br>Adjust to pH 7.5 prior to use. This requires approximately 25 ml of 1M NaOH per liter.<br><br><b>15/10 medium</b><br><br>Growth of competent TOP10 cells in Example 2 of the Bloom05 patent is performed in 15/10 broth, which is similar to SOB:</p> | SOB medium is also available dry premixed from Difco, 0443-17.<br>Adjust to pH 7.5 prior to use. This requires approximately 25 ml of 1M NaOH per liter.<br><br><b>15/10 medium</b><br><br>Growth of competent TOP10 cells in Example 2 of the Bloom05 patent is performed in 15/10 broth, which is similar to SOB:</p> | ||
Line 462: | Line 463: | ||
<p> | <p> | ||
<br>Materials | <br>Materials | ||
− | |||
<li>D-Luciferin free acid</li> | <li>D-Luciferin free acid</li> | ||
<li>ATP</li> | <li>ATP</li> | ||
Line 469: | Line 469: | ||
<li>Lysozyme</li> | <li>Lysozyme</li> | ||
<li>10 mM Tris-HCl</li> | <li>10 mM Tris-HCl</li> | ||
− | |||
</p> | </p> | ||
<p> | <p> | ||
− | Lysis Buffer | + | <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. | <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> | ||
<p> | <p> | ||
− | Reagent Solution | + | <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. | <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> | ||
<p> | <p> | ||
− | 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 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. | <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 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> |
Revision as of 18:01, 16 September 2015