Oscarliu117 (Talk | contribs) |
Oscarliu117 (Talk | contribs) |
||
Line 17: | Line 17: | ||
<a class="list-group-item list-group-item-action" href="#list-item-1">PCR</a> | <a class="list-group-item list-group-item-action" href="#list-item-1">PCR</a> | ||
<a class="list-group-item list-group-item-action" href="#list-item-2">Plasmid Construction</a> | <a class="list-group-item list-group-item-action" href="#list-item-2">Plasmid Construction</a> | ||
− | <a class="list-group-item list-group-item-action" href="#list-item-3">PCR Clean-Up & Gel Extraction</a> | + | <a class="list-group-item list-group-item-action" href="#list-item-3">PCR Clean-Up & Gel |
+ | Extraction</a> | ||
<a class="list-group-item list-group-item-action" href="#list-item-4">Plasmid Extraction</a> | <a class="list-group-item list-group-item-action" href="#list-item-4">Plasmid Extraction</a> | ||
− | <a class="list-group-item list-group-item-action" href="#list-item-5">DNS reductive sugar measurement</a> | + | <a class="list-group-item list-group-item-action" href="#list-item-5">DNS reductive sugar |
+ | measurement</a> | ||
<a class="list-group-item list-group-item-action" href="#list-item-6">Plasmid Construction</a> | <a class="list-group-item list-group-item-action" href="#list-item-6">Plasmid Construction</a> | ||
<a class="list-group-item list-group-item-action" href="#list-item-7">SS</a> | <a class="list-group-item list-group-item-action" href="#list-item-7">SS</a> | ||
<a class="list-group-item list-group-item-action" href="#list-item-8">Plasmid Construction</a> | <a class="list-group-item list-group-item-action" href="#list-item-8">Plasmid Construction</a> | ||
− | <a class="list-group-item list-group-item-action" href="#list-item-9"> | + | <a class="list-group-item list-group-item-action" href="#list-item-9">Total Solution</a> |
<a class="list-group-item list-group-item-action" href="#"><i class="fa fa-arrow-up fa-1x" | <a class="list-group-item list-group-item-action" href="#"><i class="fa fa-arrow-up fa-1x" | ||
aria-hidden="true"></i></a> | aria-hidden="true"></i></a> | ||
Line 520: | Line 522: | ||
<a class="btn col-md-12" data-toggle="collapse" href="#DNS" role="button" aria-expanded="false" | <a class="btn col-md-12" data-toggle="collapse" href="#DNS" role="button" aria-expanded="false" | ||
aria-controls="multiCollapseExample1"> | aria-controls="multiCollapseExample1"> | ||
− | DNS | + | DNS Reductive Sugar Measurement |
<i class="fa fa-arrow-down fa-10" aria-hidden="true"></i> | <i class="fa fa-arrow-down fa-10" aria-hidden="true"></i> | ||
</a> | </a> | ||
Line 580: | Line 582: | ||
</div> | </div> | ||
− | <div id="list-item- | + | <div id="list-item-6"> |
</br></br></br></br> | </br></br></br></br> | ||
Line 593: | Line 595: | ||
<div class="collapse multi-collapse" id="DNS"> | <div class="collapse multi-collapse" id="DNS"> | ||
+ | </div> | ||
+ | |||
+ | </div> | ||
+ | |||
+ | <div id="list-item-7"> | ||
+ | |||
+ | </br></br></br></br> | ||
+ | <div class="row"> | ||
+ | <a class="btn col-md-12" data-toggle="collapse" href="#DNS" role="button" aria-expanded="false" | ||
+ | aria-controls="multiCollapseExample1"> | ||
+ | DNS reductive sugar measurement | ||
+ | <i class="fa fa-arrow-down fa-10" aria-hidden="true"></i> | ||
+ | </a> | ||
+ | </div> | ||
+ | |||
+ | <div class="collapse multi-collapse" id="DNS"> | ||
+ | |||
+ | </div> | ||
+ | |||
+ | </div> | ||
+ | |||
+ | <div id="list-item-8"> | ||
+ | |||
+ | </br></br></br></br> | ||
+ | <div class="row"> | ||
+ | <a class="btn col-md-12" data-toggle="collapse" href="#COO" role="button" aria-expanded="false" | ||
+ | aria-controls="multiCollapseExample1"> | ||
+ | DNS reductive sugar measurement | ||
+ | <i class="fa fa-arrow-down fa-10" aria-hidden="true"></i> | ||
+ | </a> | ||
+ | </div> | ||
+ | |||
+ | <div class="collapse multi-collapse" id="COO"> | ||
+ | <p class="pcontent"> | ||
+ | <h3>Materials:</h3> | ||
+ | pH meter, enzyme samples, magnetic stirrer, saturated CO2 solution, 20 mM Tris-HCl | ||
+ | buffer (pH8.3), 20mL borosilicate glass vial | ||
+ | |||
+ | |||
+ | <h3>Method:</h3> | ||
+ | |||
+ | Saturated CO2 solution preparation</br></br> | ||
+ | Dissolve gaseous CO2 into deionized water (on ice) until it is saturated. (At least | ||
+ | 30 min)</br></br> | ||
+ | |||
+ | 20 mM Tris HCl buffer (pH8.3) preparation</br></br> | ||
+ | <li>Dissolve 121.14 g Tris in 800 ml deionized water.</li></br> | ||
+ | <li>Add a pH meter into the solution to observe the pH.</li></br> | ||
+ | <li>Slowly add concentrated hydrochloric acid (HCl) solution to reduce the pH to | ||
+ | 8.3. Be careful not to add too much at a time, since the pH will change | ||
+ | rapidly.</li></br> | ||
+ | <li>Once the desired pH has been reached, top up the solution to 1 L using | ||
+ | deionized water.</li></br> | ||
+ | |||
+ | Performing Blank Reaction</br></br> | ||
+ | <li>Add 9 mL ice-cold Tris−HCl (20 mM, pH8.3) buffer into a 20 mL borosilicate | ||
+ | glass vial with further incubation at 0 °C with stirring.</li></br> | ||
+ | <li>Add 6 mL of ice-cold CO2-saturated solution immediately into the vial.</li></br> | ||
+ | <li>Record the time course, T<sub>0</sub> (in sec) of pH decrease from 8.3 to | ||
+ | 6.3. The pH meter must be preset to 0°C and calibrated.</li></br> | ||
+ | |||
+ | Performing Test-Sample Reaction</br></br> | ||
+ | <li>Mix 9 mL ice-cold Tris−HCl (20 mM, pH8.3) buffer and 0.2 mL enzyme, transfer | ||
+ | the mixture to a 20 mL borosilicate glass vial with further incubation at 0 °C | ||
+ | with stirring.</li></br> | ||
+ | <li>Add 6 mL of ice-cold CO2-saturated solution immediately into the vial. | ||
+ | <li>Record the time course, T (in sec) of pH decrease from 8.3 to 6.3. The pH | ||
+ | meter must be preset to 0°C and calibrated.</li></br> | ||
+ | <li>Calculate CA activity using a Wilbur–Anderson unit (WAU) per milliliter of | ||
+ | sample.</li></br> | ||
+ | |||
+ | </p> | ||
+ | </div> | ||
+ | |||
+ | </div> | ||
+ | |||
+ | <div id="list-item-9"> | ||
+ | |||
+ | </br></br></br></br> | ||
+ | <div class="row"> | ||
+ | <a class="btn col-md-12" data-toggle="collapse" href="#TLT" role="button" aria-expanded="false" | ||
+ | aria-controls="multiCollapseExample1"> | ||
+ | Total Solution | ||
+ | <i class="fa fa-arrow-down fa-10" aria-hidden="true"></i> | ||
+ | </a> | ||
+ | </div> | ||
+ | |||
+ | <div class="collapse multi-collapse" id="TLT"> | ||
+ | <p class="pcontent"> | ||
+ | <ol> | ||
+ | <li>Preculture the bacteria overnight by picking up a colony in 4ml LB with | ||
+ | antibiotic.</li></br> | ||
+ | |||
+ | <li>Culture the bacteria in 30 ml LB by adding 1/100 of bacteria , 1/2000 | ||
+ | kanamycin, 1/4000 chloramphenicol to log phase( about 3 hr).</li></br> | ||
+ | |||
+ | <li>Centrifuge them in 22℃ ,3200xg for 5 minutes , then refresh by the M9 | ||
+ | medium with 4%xylose and 0.1%LB.</li></br> | ||
+ | |||
+ | <li>Culture the bacteria for 24 hr in both O2 and 5%CO2 incubator , and test | ||
+ | the O.D. value at every 6 hours.</li></br> | ||
+ | </ol> | ||
+ | </p> | ||
</div> | </div> | ||
Revision as of 12:54, 29 September 2018