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| - | <h4><font size="4" face="calibri"><b>Competent cell</b></font>
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| - | <font size="3" face="calibri">
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| - | <ol>
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| - | <li>Culture DH5-alpha in a glass test tube with 3CC LB at 37°C for 12 hours.
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| - | <li>Add 200 ul bacterium into a 500cc conical flask with 50 ml LB inside. Culture at 37°C until OD600 reaches 0.2
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| - | <li>3. Distribute the bacterium from the conical flask into a 50cc Centrifuge and centrifuge at 4000rpm, 15 minutes at 4 °C
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| - | <li>Remove supernatant and add in 16 ml CaCl2 (100mM)
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| - | <li>Centrifuge at 4000rpm, 15 minutes at 4 °C
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| - | <li>Remove supernatant and add 3 ml CaCl2 (100mM)
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| - | <li>Centrifuge at 4000rpm, 15 minutes at 4 °C
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| - | <li>Remove supernatant and add 3 ml CaCl2 (100mM)
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| - | <li>Place on ice and put in 4 °C cold room for an hour.
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| - | <li>Cut tip and add 1 ml Glycerol (Takes up 25% of the total volume)
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| - | <li>Transfer 200 ul into each eppendorf ( This step should be done quickly and taken to -80°C ASAP)<br>* Can use dry ice or ice with alcohol.
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| - | <li>Plating the next day to test CFU (colony frequency unit)
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| - | </ol>
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| - | </font>
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| - | </h4>
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| - | <hr>
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| - | <h4>
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| - | <font size="4" face="calibri"><b>Competent CFU test - Transformation-E.coli<br><br><dl><dd>Test competent cell CFU the day before</dl></b></font>
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| - | <font size="3" face="calibri">
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| - | <ol>
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| - | <li>Defrost the 200 ul competent cell on ice.
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| - | <li>Add 10 ng plasmid into the 200 ul competent cell.
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| - | <li>Place it on ice for 30 minutes
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| - | <li>Heat shock in water bath at 42℃ for 1 and a half minute then place it on ice for 5 minutes.
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| - | <li>Add in 800 ul LB and place it in the 37℃ incubator for 1 hour.
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| - | <li>Test the frequency
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| - | <li>Incubate at 37℃for 12~16 hours
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| - | <li>Select colony and check via quick- screening.
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| - | </ol>
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| - | </font>
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| - | </h4>
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| - | <hr>
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| - | <h4>
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| - | <font size="4" face="calibri"><b>Glycerol Stock<br><br><dl><dd>Stock<br>1.8 % NaCl<br>50 % glycerol</dl></b></font>
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| - | <font size="3" face="calibri">
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| - | <ol>
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| - | <li>Transfer 500 µl Bacterium (with LB) into freezing tube.
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| - | <li>Add 500 µl stock (Bacterium::Stock = 1:1)
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| - | <li>Store at -80℃<dl><dd>*Rest of the bacterium should be autoclaved and disposed.</dl>
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| - | </ol>
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| - | </font>
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| - | </h4>
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| - | <hr>
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| - | <h4>
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| - | <font size="4" face="calibri"><b>Digestion</b></font>
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| - | <font size="3" face="calibri">
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| - | <ol>
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| - | <li>Do a nanodrop test on the plasmid we want to cleave.
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| - | <li>Add in the materials as follows:
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| - | <br>
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| - | <br>
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| - | <dl>
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| - | <dd>Plasmid or DNA X ul (Total of 10ug therefore add approximately >5ul)<br>10x buffer 10 ul<br>RE 1 u ( 1 ul per restriction enzyme)<br>dd water add to 100 ul<br>total 100 ul
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| - | <br></dl>
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| - | Let in react in 37°C water bath.
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| - | <br>
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| - | <br>
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| - | *An electrophoresis can be done to double check if we have successfully cleaved our plasmid. Only one band should be seen.
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| - | </ol>
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| - | </font>
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| - | </h4>
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| - |
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| - | <hr>
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| - | <h4>
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| - | <div style="float:left; width:35%">
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| - | <font size="4" face="calibri"><b>Ligation</b></font>
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| - | <font size="3" face="calibri">
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| - | <ol>
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| - | <li>Pepare cocktail<br><br>
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| - | <dl>
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| - | <dd>
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| - | <table style="border:6px ridge rbg(109,2,107); height: 100px ; background-color: white ; width: 200px ;" align="left" cellpadding="0" cellspacing="5" frame="border" rules="all">
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| - | <tbody>
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| - | <tr>
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| - | <td>
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| - | <dl>
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| - | <dd>
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| - | Insert DNA
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| - | </td>
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| - | <td>
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| - | <dd>
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| - | 5µl
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| - | </td>
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| - | </tr>
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| - | </dl>
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| - | <tr>
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| - | <td>
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| - | <dl>
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| - | <dd>
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| - | plasmid
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| - | </td>
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| - | <td>
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| - | <dd>
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| - | 1µl
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| - | </td>
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| - | </tr>
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| - | </dl>
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| - | <tr>
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| - | <td>
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| - | <dl>
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| - | <dd>
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| - | 10X buffer
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| - | </td>
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| - | <td>
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| - | <dd>
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| - | 1µl
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| - | </td>
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| - | </tr>
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| - | </dl>
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| - | <tr>
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| - | <td>
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| - | <dl>
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| - | <dd>
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| - | ligase
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| - | </td>
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| - | <td>
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| - | <dd>
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| - | 1µl
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| - | </td>
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| - | </tr>
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| - | </dl>
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| - | <tr>
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| - | <td>
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| - | <dl>
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| - | <dd>
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| - | 10mM ATP
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| - | </td>
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| - | <td>
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| - | <dd>
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| - | 1µl
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| - | </td>
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| - | </tr>
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| - | </dl>
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| - | <tr>
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| - | <td>
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| - | <dl>
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| - | <dd>
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| - | ddH2O
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| - | </td>
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| - | <td>
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| - | <dd>
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| - | 1µl
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| - | </td>
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| - | </tr>
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| - | </dl>
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| - | <tr>
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| - | <td>
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| - | <dl>
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| - | <dd>
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| - | total
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| - | </td>
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| - | <td>
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| - | <dd>
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| - | 10µl
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| - | </td>
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| - | </tr>
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| - | </dl>
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| - | </tbody>
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| - | </table>
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| - |
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| - | </div>
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| - | <br>
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| - | <br>
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| - | <br>
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| - | <br>
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| - | <br>
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| - | <dl>
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| - | <dd>Place it in 22℃ dry bath for 1hr/overnight
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| - | <br>
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| - | <br>
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| - | <dd>* Insert DNA : Plasmid = M ole ratio 5:1 or 3:1
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| - | <br>
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| - | <br>
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| - | <dd><img src="https://static.igem.org/mediawiki/2013/d/dc/Protocol.png">
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| - | </dl>
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| - | <br>
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| - | <br>
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| - | <br>
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| - | <br>
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| - | <br>
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| - | </h4>
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| - |
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| - | <hr>
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| - |
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| - | <h4>
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| - | <font size="4" face="calibri"><b>Gel Extraction Kit</b></font>
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| - | <font size="3" face="calibri">
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| - | <ol>
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| - | <li>Excise the gel slice containing the DNA band with a clean, sharp scalpel.
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| - | <li>Weigh the gel slice. Add 1–2 volumes of diffusion buffer to 1 volume of gel.
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| - | <li>Incubate at 65°C for 10 min.
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| - | <li>Centrifuge the sample for 1 min.
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| - | <li>Carefully remove the supernatant using a pipet or a drawn-out Pasteur pipet. Pass the supernatant through a disposable plastic column or a syringe containing either a Whatman GF/C filter or packed, siliconized glass wool to remove any residual polyacrylamide.
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| - | <li>Determine the volume of the recovered supernatant.
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| - | <li>Add 3 volumes of Buffer QG to 1 volume of supernatant and mix. Check that the color of the mixture is yellow.
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| - | <li>Place a QIAquick Spin Column in a provided 2 ml collection tube.
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| - | <li>To bind DNA, apply the sample to the QIAquick Spin Column and centrifuge for 1 min.
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| - | <li>Discard flow-through and place QIAquick Spin Column back into the same collection tube.
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| - | <li>To wash, add 0.75 ml Buffer PE to column and centrifuge for 1 min.
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| - | <li>Discard flow-through and place QIAquick Spin Column back in the same tube.Centrifuge column for an additional 1 min at maximum speed.
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| - | <li>Place QIAquick Spin Column into a clean 1.5 ml microcentrifuge tube.
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| - | <li>To elute DNA, add 50 μl TE Buffer or water to the center of the QIAquick Spin Column and centrifuge for 1 min.
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| - | </ol>
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| - | </h4>
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| - |
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| - | <hr>
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| - | <h4>
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| - | <font size="4" face="calibri"><b>Plasmid Purification</b></font>
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| - | <font size="3" face="calibri">
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| - | <ol>
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| - | <li>Pellet of the cells by centrifugation for 10 min at 12000rpm.
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| - | <li>Pour off the supernatant and remove excess media.
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| - | <li>Completely resuspend the cell pellet in 100 μl of Solution I by pipetting or vortex.
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| - | <li>Add 200 μl freshly prepared Solution II(2% SDS:0.4N NaOH = 1:1)and mix by inverting the tube 5-7 times, then let stand for 5 min.
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| - | <li>Add 150 μl Solution III and mix by inverting the tube ten times, put on ice for 5 min, then centrifuge at 12000rpm, 10 min at 4℃.
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| - | <li>Transfer the supernatant into a new eppendorf.
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| - | <li>Add 3 μl RNaseA, and put in 37℃ water bath for 30 min.
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| - | <li>Add 1 volume phenol/chloroform, inverting gently.
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| - | <li>Centrifuge at 12000rpm, 10 min at 4℃.
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| - | <li>Transfer the supernatant into a new eppendorf.
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| - | <li>Add 1 volume chloroform, inverting gently.
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| - | <li>Centrifuge at 12000rpm, 10 min at 4℃.
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| - | <li>Transfer the supernatant into a new eppendorf.
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| - | <li>Add 1 volume 95% ethanol, and put it on room temperature for 20 min.
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| - | <li>Centrifuge at 12000rpm, 10 min at 4℃.
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| - | <li>Air dry the pellet for 30 min or overnight.
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| - | <li>Add 30 μl TE buffer to elute.
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| - | </ol>
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| - | </h4>
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