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index 688c062b97e114c6139a09c97d09afb09adbcbc3..78bcbdc679746a04538228f10c46aebb857c1b62 100644
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@@ -157,6 +157,177 @@
</li>
</ol>
</details>
+
+ <details>
+ <summary>
+ <h2>Double digestion by restriction enzyme</h2>
+ </summary>
+ <ol>
+ <li>
+ Mix as follows and incubate at 37°C for 2 hours.
+ <table>
+ <tr>
+ <th style="background-color: black; color: white;">compounds</th>
+ <th style="background-color: black; color: white;">μL</th>
+ </tr>
+ <tr>
+ <td>CutSmart Buffer x10</td>
+ <td>5</td>
+ </tr>
+ <tr>
+ <td>vector</td>
+ <td>6</td>
+ </tr>
+ <tr>
+ <td>Enzymes</td>
+ <td>1 each</td>
+ </tr>
+ <tr>
+ <td>DW</td>
+ <td>X</td>
+ </tr>
+ </table>
+ </li>
+ <li>
+ Electrophorese with the uncut vector to confirm that it was correctly cleaved (only one band in the middle of the two bands of the uncut vector), and then cut the gel out and extract the gel.
+ </li>
+ </ol>
+ </details>
+
+ <details>
+ <summary>
+ <h2>Gibson Assembly</h2>
+ </summary>
+ <ol>
+ <li>
+ Mix as follows and incubate at 50°C for 30 minutes.
+ <table>
+ <tr>
+ <th style="background-color: black; color: white;">compounds</th>
+ <th style="background-color: black; color: white;">μL</th>
+ </tr>
+ <tr>
+ <td>Master Mix</td>
+ <td>3</td>
+ </tr>
+ <tr>
+ <td>Vector</td>
+ <td>1</td>
+ </tr>
+ <tr>
+ <td>inserts</td>
+ <td>2 each</td>
+ </tr>
+ </table>
+ </li>
+ </ol>
+ </details>
+
+ <details style="border: 2px solid #999999; border-radius: 10px; padding: 10px;">
+ <summary>
+ <h2>Transformation</h2>
+ </summary>
+ <ol>
+ <li>
+ Place competent cells on ice for 15 minutes to thaw in a microtube. During this time, set the heat block to 56°C.
+ </li>
+ <li>
+ Add all of the Gibson Assembly reaction solution to the competent cells.
+ </li>
+ <li>
+ Conduct heat shock placing the cells. 0°C for 5 min, 56°C for 35 sec, and 0°C for 5 min.
+ </li>
+ <li>
+ Add 100 μL of antibiotic-free liquid LB medium, and recover at 37°C for 1 hour.
+ </li>
+ <li>
+ Spread the cells on antibiotic-added LB agar medium and incubate at 37°C overnight.
+ </li>
+ </ol>
+ </details>
+
+ <details style="border: 2px solid #999999; border-radius: 10px; padding: 10px;">
+ <summary>
+ <h2>Plasmid extraction</h2>
+ </summary>
+ <ol>
+ <li>
+ Poke One colony on plate with a sterilized pipette tip.
+ </li>
+ <li>
+ Put the tip into antibiotic-added liquid LB medium and incubate with shaking until the medium became turbid.
+ </li>
+ <li>
+ Extract plasmids according to FastGene Plasmid Mini.
+ </li>
+ <li>
+ Measure DNA concentration using Nanodrop 2000.
+ </li>
+ </ol>
+ </details>
+
+ <h2>Culturing, Compounds Analysis</h2>
+ <hr>
+
+ <details style="border: 2px solid #999999; border-radius: 10px; padding: 10px;">
+ <summary>
+ <h2>Pre-culture and main culture</h2>
+ </summary>
+ <ol>
+ <li>
+ Add one drop of the inoculum to a 15 mL test tube containing antibiotic-added liquid LB medium and incubate at 37°C with shaking.
+ </li>
+ <li>
+ When the culture medium became turbid, transfer it to a baffled Erlenmeyer flask containing 100 mL of antibiotic-added liquid LB medium and incubate at 37°C, 120 rpm for 3 hours with shaking.
+ </li>
+ </ol>
+ </details>
+
+ <details style="border: 2px solid #999999; border-radius: 10px; padding: 10px;">
+ <summary>
+ <h2>Substance Synthesis</h2>
+ </summary>
+ <ol>
+ <li>
+ Centrifuge the culture medium and concentrate the bacteria to make a pellet. Collect the supernatant in an appropriate container and autoclaved to sterilize.
+ </li>
+ <li>
+ Add saline solution to the pellet, vortex to suspend it, and measure OD.
+ </li>
+ <li>
+ Add saline so that the OD was 10.
+ </li>
+ <li>
+ To a 50 mL Falcon, add 4.5 mL of medium for material synthesis and 0.5 mL of E. coli suspension. After that, shake for the specified time.
+ </li>
+ </ol>
+ </details>
+
+ <details style="border: 2px solid #999999; border-radius: 10px; padding: 10px;">
+ <summary>
+ <h2>Solvent extraction and LC/MS of CDP</h2>
+ </summary>
+ <ol>
+ <li>
+ To 3 mL of the reaction solution, add 3 mL of ethyl acetate, shake well, and centrifuge at 10,000 rpm for 1 minute.
+ </li>
+ <li>
+ Reserve 2.5 mL of the oil layer, add 2.5 mL of ethyl acetate to the remainder, and repeat the same mixing, separation, and preparative procedure twice.
+ </li>
+ <li>
+ Evaporate 7.5 mL of ethyl acetate solution of the sample in a centrifugal evaporator (Sakuma Seisakusho EC100) at reduced pressure.
+ </li>
+ <li>
+ Dissolved the remaining solid in 100 μL of methanol, transfer it to a micro tube, and centrifuge at 15,000 rpm for 10 min. collect 40 μL of the supernatant carefully and place in an ampoule to make a sample.
+ </li>
+ <li>
+ Conduct LC/MS analysis (SHIMADZU LCMS-8045) using the following settings<br>
+ Organic solvent: acetonitrile<br>
+ Inorganic solvent: formic acid<br>
+ time: 26 minutes
+ </li>
+ </ol>
+ </details>
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