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− | background-color: # | + | background-color: #e6faff; |
font-family: roboto; | font-family: roboto; | ||
} | } | ||
+ | |||
+ | section { | ||
+ | font-family: roboto; | ||
+ | margin:auto; | ||
+ | position: relative; | ||
+ | width: 85%; | ||
+ | height: auto; | ||
+ | } | ||
</style> | </style> | ||
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<body> | <body> | ||
− | + | <section id="section01"> | |
− | <section> | + | <div class="splash" id="mainsplash"> |
+ | <div id="banner"> | ||
+ | <img src="https://static.igem.org/mediawiki/2017/4/4f/Notebook_protocal.png" width="100%" height="auto" class=" igem-logo"> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="column full_size"> | ||
<br><br> | <br><br> | ||
− | <p style="font-family: | + | <p style="font-family: roboto;font-size:150%;">Below are the protocols that we used in our project. Click the boxes for more detail! </p> |
<br> | <br> | ||
+ | <div class="some-padding"></div> | ||
+ | <div class="some-padding"></div> | ||
+ | <div class="some-padding"></div> | ||
+ | <div class="some-padding"></div> | ||
+ | <div class="panel-group" id="accordion" role="tablist" aria-multiselectable="true"> | ||
+ | <div class="panel panel-default"> | ||
+ | <div class="panel-heading" role="tab" id="P0"> | ||
+ | <h4 class="panel-title"> | ||
+ | <a role="button" data-toggle="collapse" data-parent="#accordion" href="#P0-collapse" aria-expanded="false" aria-controls="P0-collapse"> | ||
+ | <div> | ||
+ | <div class="col-md-11">PCR </div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div> | ||
+ | </div> | ||
+ | </a> | ||
+ | </h4> | ||
+ | |||
+ | </div> | ||
+ | <div id="P0-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="P0"> | ||
+ | <div class="panel-body"> | ||
+ | <ol> | ||
+ | <li>Set up PCR mixture as follow :</li> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Component</th> | ||
+ | <th>Volume</th> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Phusion DNA Polymerase</td> | ||
+ | <td>0.5 μl </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>5X Phusion Green HF Buffer </td> | ||
+ | <td>10 μl </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>10mM dNTPs </td> | ||
+ | <td>1 μl </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Forward Primer </td> | ||
+ | <td>2.5 μl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Reverse Primer </td> | ||
+ | <td>2.5 μl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>DNA template </td> | ||
+ | <td>1 μl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>DNase-free ddH2O </td> | ||
+ | <td>32.5μl</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <table> | ||
+ | <tr> | ||
+ | <th>Procedures</th> | ||
+ | <th>Temperature (°C)</th> | ||
+ | <th>Duration</th> | ||
+ | <th>Cycle</th> | ||
+ | </tr> | ||
+ | <li>Set up thermocycler for PCR reaction as follow:</li> | ||
+ | <tr> | ||
+ | <td>Initial denaturation </td> | ||
+ | <td>98</td> | ||
+ | <td>30 s</td> | ||
+ | <td>1</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Denaturation </td> | ||
+ | <td>98</td> | ||
+ | <td>5 s</td> | ||
+ | <td>1</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Annealing </td> | ||
+ | <td>64</td> | ||
+ | <td>30 s</td> | ||
+ | <td>25</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Final extension </td> | ||
+ | <td>72</td> | ||
+ | <td>5 mins</td> | ||
+ | <td>1</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Sample keping</td> | ||
+ | <td> 4 </td> | ||
+ | <td> ∞ </td> | ||
+ | <td> 1</td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | <li>Add the other Component in the tube. If the added volume is less, pipetting up and down | ||
+ | is necessary. </li> | ||
+ | <li>Mix reagents in tubes by pipetting the solution up and down slowly. </li> | ||
+ | <li>Quick spin the PCR tube to ensure the mixture is in the bottom of the tube. </li> | ||
+ | <li>Put it in the thermocycler (PCR machine) and set the cycle information. </li> | ||
+ | <li>Start the cycle and wait till it is finished. </li> | ||
+ | </ol> | ||
+ | <a href="https://www.neb.com/protocols/1/01/01/pcr-protocol-m0530">Click here for the manufacturer's maual</a> | ||
+ | |||
+ | </p> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
<div class="panel-group" id="accordion" role="tablist" aria-multiselectable="true"> | <div class="panel-group" id="accordion" role="tablist" aria-multiselectable="true"> | ||
<div class="panel panel-default"> | <div class="panel panel-default"> | ||
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<a role="button" data-toggle="collapse" data-parent="#accordion" href="#P1-collapse" aria-expanded="false" aria-controls="P1-collapse"> | <a role="button" data-toggle="collapse" data-parent="#accordion" href="#P1-collapse" aria-expanded="false" aria-controls="P1-collapse"> | ||
<div> | <div> | ||
− | <div class="col-md-11"> | + | <div class="col-md-11">Gel electrophoresis</div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div> |
</div> | </div> | ||
</a> | </a> | ||
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<ol> | <ol> | ||
− | <li> | + | <li>Prepare your 1% gel by using 0.5g of agarose in 50 ml of TAE buffer.</li> |
− | <li>Add | + | <li>Add 2.5ul of Ethidium Bromide before you pour your gel into the chamber. |
− | + | <li>Mix 5ul of DNA with 1ul of loading buffer by pipetting up and down a couple of times. </li> | |
− | <li> | + | <li>Load your samples and appropriate marker into your wells</li> |
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | <li> | + | |
<li>Incubate at 37°C and 250 rpm for 60-120 minutes.</li> | <li>Incubate at 37°C and 250 rpm for 60-120 minutes.</li> | ||
− | <li> | + | <li>Apply 130 volts to the chamber for 20 mins. </li> |
− | <li> | + | <li>Check your gel using a transilluminator or other UV emitting device.</li> |
<li>Re-suspend cells by light vortexing</li> | <li>Re-suspend cells by light vortexing</li> | ||
<li>Plate resuspended cells as above</li> | <li>Plate resuspended cells as above</li> | ||
<li>Incubate overnight at 37°C with plates upside down.</li> | <li>Incubate overnight at 37°C with plates upside down.</li> | ||
</ol> | </ol> | ||
+ | <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4846332/">Click here for the manufacturer's maual</a> | ||
</div> | </div> | ||
</div> | </div> | ||
</div> | </div> | ||
+ | <div class="panel-group" id="accordion" role="tablist" aria-multiselectable="true"> | ||
+ | <div class="panel panel-default"> | ||
+ | <div class="panel-heading" role="tab" id="P2"> | ||
+ | <h4 class="panel-title"> | ||
+ | <a role="button" data-toggle="collapse" data-parent="#accordion" href="#P2-collapse" aria-expanded="false" aria-controls="P2-collapse"> | ||
+ | <div> | ||
+ | <div class="col-md-11">DNA Gel Purification</div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div> | ||
+ | </div> | ||
+ | </a> | ||
+ | </h4> | ||
+ | </div> | ||
+ | <div id="P2-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="P2"> | ||
+ | <div class="panel-body"> | ||
− | < | + | <ol> |
− | < | + | <li>Excise the DNA fragment from the agarose gel with a clean, sharp scalpel. Minimize the size of the gel slice by trimming off extra agarose gel.</li> |
+ | <li>Weigh the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume of gel (300 μl QG to 100 mg gel).</li> | ||
+ | <li>Incubate the tube at 60°C until the gel completely dissolved. </li> | ||
+ | <li> For DNA fragments <500 bp and >4 kb, add 1 gel volume of isopropanol to the sample and mix the reagents well. </li> | ||
+ | <li>o bind DNA, apply 800 μl sample to the QIAquick column, and centrifuge for 1 min. </li> | ||
+ | <li>Discard flow-through and place QIAquick column back in the same collection tube. It can be reused to reduce plastic waste.</li> | ||
+ | <li>Add 0.75 ml of Buffer PE to wash the QIAquick column, and centrifuge for 1 min. </li> | ||
+ | <li>Add 30 μl of distilled water to elute DNA.</li> | ||
+ | </ol> | ||
+ | <a href=" http://eng.intronbio.com/PROTO-PDF/mega-spin.pdf">Click here for the manufacturer's maual</a> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
<div class="panel-group" id="accordion" role="tablist" aria-multiselectable="true"> | <div class="panel-group" id="accordion" role="tablist" aria-multiselectable="true"> | ||
<div class="panel panel-default"> | <div class="panel panel-default"> | ||
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<a role="button" data-toggle="collapse" data-parent="#accordion" href="#P3-collapse" aria-expanded="false" aria-controls="P3-collapse"> | <a role="button" data-toggle="collapse" data-parent="#accordion" href="#P3-collapse" aria-expanded="false" aria-controls="P3-collapse"> | ||
<div> | <div> | ||
− | <div class="col-md-11"> | + | <div class="col-md-11">Restriction digestion</div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div> |
</div> | </div> | ||
</a> | </a> | ||
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<div class="panel-body"> | <div class="panel-body"> | ||
<ol> | <ol> | ||
− | <li>Prepare the | + | <li>Prepare the reagents as follow:</li> |
<table> | <table> | ||
<tr> | <tr> | ||
− | <th> | + | <th>Reaction Components</th> |
− | <th> | + | <th>Volume</th> |
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td> | + | <td>DNA template</td> |
− | <td>0. | + | <td>0.5 μg</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td> | + | <td>10X Buffer</td> |
− | <td> | + | <td>2.5 μl</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td> | + | <td>Enzyme</td> |
− | <td> | + | <td>2 μl</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td> | + | <td>Nuclease-Free Water to final colume of:</td> |
− | <td> | + | <td> 25 μl</td> |
</tr> | </tr> | ||
− | + | </table> | |
− | + | <li>Pipette the solution up and down to ensure all reagents are mixed well.</li> | |
− | + | <li>Place the reaction mixture at 37 oC incubation or dry bath for 2-4 hours. </li> | |
− | + | <li>Purify the DNA by PCR purification kit/gel extraction kit for downstream process. </li> | |
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | <li> | + | |
− | <li> | + | |
</ol> | </ol> | ||
+ | <a href=" https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0012178_XbaI_10_UuL_5X1500U_UG.pdf">Click here for the manufacturer's maual</a> | ||
</p> | </p> | ||
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</div> | </div> | ||
</div> | </div> | ||
− | |||
− | |||
− | |||
<div class="panel-group" id="accordion" role="tablist" aria-multiselectable="true"> | <div class="panel-group" id="accordion" role="tablist" aria-multiselectable="true"> | ||
<div class="panel panel-default"> | <div class="panel panel-default"> | ||
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<div class="panel-body"> | <div class="panel-body"> | ||
<ol> | <ol> | ||
− | <li>Prepare the | + | <li>Prepare the reaction mixture as follow:</li> |
<table> | <table> | ||
<tr> | <tr> | ||
Line 166: | Line 289: | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td> | + | <td>DNA </td> |
<td>0.2M KCl/HCl</td> | <td>0.2M KCl/HCl</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>DNA template</td> | <td>DNA template</td> | ||
− | <td> | + | <td>10-200 ng</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td> | + | <td>10XT4 DNA Ligase Reaction Buffer</td> |
− | <td>2 | + | <td>2 μl</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td> | + | <td>T4 DNA Ligase</td> |
− | <td> | + | <td>1 μl</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>Water</td> | <td>Water</td> | ||
− | <td>Top up to | + | <td>Top up to 20 μl</td> |
</tr> | </tr> | ||
</table> | </table> | ||
− | <li> | + | <li>Allow the ligation to take place a 22-25°C for 10 minutes.</li> |
− | <li> | + | <li>Send 5 μl of the ligated sample should be used for agarose gel electrophoresis to confirm whether ligation has occurred (Optional)</li> |
− | + | ||
</ol> | </ol> | ||
+ | |||
+ | <a href=" https://www.neb.com/protocols/0001/01/01/dna-ligation-with-t4-dna-ligase-m0202">Click here for the manufacturer's maual</a> | ||
</p> | </p> | ||
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<div class="some-padding"></div> | <div class="some-padding"></div> | ||
<div class="some-padding"></div> | <div class="some-padding"></div> | ||
− | |||
<div class="panel-group" id="accordion" role="tablist" aria-multiselectable="true"> | <div class="panel-group" id="accordion" role="tablist" aria-multiselectable="true"> | ||
<div class="panel panel-default"> | <div class="panel panel-default"> | ||
− | <div class="panel-heading" role="tab" id=" | + | <div class="panel-heading" role="tab" id="P9"> |
<h4 class="panel-title"> | <h4 class="panel-title"> | ||
− | <a role="button" data-toggle="collapse" data-parent="#accordion" href="# | + | <a role="button" data-toggle="collapse" data-parent="#accordion" href="#P9-collapse" aria-expanded="false" aria-controls="P9-collapse"> |
<div> | <div> | ||
− | <div class="col-md-11"> | + | <div class="col-md-11">Preparation of competnet cells </div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div> |
</div> | </div> | ||
</a> | </a> | ||
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</div> | </div> | ||
− | <div id=" | + | <div id="P9-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="P9"> |
<div class="panel-body"> | <div class="panel-body"> | ||
− | <ol> | + | <ol> |
− | <li>Prepare the | + | <li>Prepare the Ca/glycerol buffer as follow and flow through 0.22 μM filter.</li> |
<table> | <table> | ||
<tr> | <tr> | ||
− | <th> | + | <th>Composition</th> |
<th>Volume</th> | <th>Volume</th> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td> | + | <td>0.6 M CaCl2 </td> |
− | <td> | + | <td>10 ml </td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td> | + | <td>0.5 M PIPES pH7.0 </td> |
− | <td> | + | <td>2 ml </td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td> | + | <td>Glycerol </td> |
− | <td> | + | <td>15 ml </td> |
</tr> | </tr> | ||
− | + | <tr> | |
− | <td> | + | <td>H2O </td> |
− | <td> | + | <td>73 ml </td> |
</tr> | </tr> | ||
− | + | <tr> | |
− | <td> | + | <td>Total </td> |
− | <td> | + | <td>100 ml </td> |
</tr> | </tr> | ||
</table> | </table> | ||
− | <li> | + | <li>Spread the bacterial cell (DH5α) for plasmid replication on the LB. <br></li> |
− | <li> | + | <li>Pick single colony in 5 ml LB culture medium and grow overnight at 37 °C by shaking at ~220 rpm. </li> |
+ | <li>Add 1ml overnight culture each to two of 100 ml flasks and grow the cell culture to achieve OD 600 = 0.25-0.4 (~ 3 h) </li> | ||
+ | <li>Transfer the cell culture (total 200 ml) to four of 50 ml sterile centrifugation tubes. </li> | ||
+ | <li>Collect the cell by centrifuging at 1000 g for 10 min at 4°C. </li> | ||
+ | <li>Gently resuspend the cell in each tube with 10 ml ice-old Ca/glycerol buffer. Keep the solution ice-cold. * Cells must remain clod for the rest of the procedures! </li> | ||
+ | <li>Collect the cell by centrifuging at 1000 g for 10 min at 4 °C. </li> | ||
+ | <li>Centrifuge at 13,000 rpm for 1 min.</li> | ||
+ | <li>Gently resuspend the cell in each tube with 1.25 ml ice-old Ca/glycerol buffer. </li> | ||
+ | <li>Centrifuge at 13,000 rpm for 1 min.</li> | ||
+ | <li>Column dring with centrifuge 13.000rpm for 1 min.</li> | ||
+ | <li>Transfer all cells to one tube. </li> | ||
+ | <li>Dispense 100 μl aliquots of competent cells into each Eppendorf.</li> | ||
+ | <li>Store at -80 °C. </li> | ||
+ | <li>Test the transformation efficiency of competent cells with antibiotics resistance plamids at different concentrations.</li> | ||
</ol> | </ol> | ||
+ | <a href=" https://www.neb.com/protocols/2012/06/21/making-your-own-chemically-competent-cells">Click here for the manufacturer's maual</a> | ||
</p> | </p> | ||
− | + | ||
− | + | </div> | |
</div> | </div> | ||
</div> | </div> | ||
− | |||
− | |||
− | |||
<div class="panel-group" id="accordion" role="tablist" aria-multiselectable="true"> | <div class="panel-group" id="accordion" role="tablist" aria-multiselectable="true"> | ||
<div class="panel panel-default"> | <div class="panel panel-default"> | ||
− | <div class="panel-heading" role="tab" id=" | + | <div class="panel-heading" role="tab" id="P5"> |
<h4 class="panel-title"> | <h4 class="panel-title"> | ||
− | <a role="button" data-toggle="collapse" data-parent="#accordion" href="# | + | <a role="button" data-toggle="collapse" data-parent="#accordion" href="#P5-collapse" aria-expanded="false" aria-controls="P5-collapse"> |
<div> | <div> | ||
− | <div class="col-md-11"> | + | <div class="col-md-11">Transformation</div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div> |
</div> | </div> | ||
</a> | </a> | ||
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</div> | </div> | ||
− | <div id=" | + | <div id="P5-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="P5"> |
<div class="panel-body"> | <div class="panel-body"> | ||
+ | |||
<ol> | <ol> | ||
− | + | <li>Thaw 100µL competent E. coli cells on ice for 10 minutes<br></li> | |
− | <li> | + | <li>Add:</li> |
− | <li> | + | <ul> |
− | <li> | + | <li>10-20 µl DNA from a ligation reaction mix OR</li> |
+ | <li>50-100ng DNA of a known plasmid </li> | ||
+ | </ul> | ||
</li> | </li> | ||
− | <li> | + | <li>Place the mixture on ice for 15 minutes. |
− | <li> | + | <li>Heat shock at exactly 42°C for 90 seconds. |
− | < | + | <li>Place on ice for 5 minutes. </li> |
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
<li>Pipette 900 µl of room temperature SOC or LB media into the mixture.</li> | <li>Pipette 900 µl of room temperature SOC or LB media into the mixture.</li> | ||
− | <li> | + | <li>Incubate at 37°C and 250 rpm for 60-120 minutes.</li> |
− | </li> | + | <li>Pellet cells at 13000rpm for 1 minutes</li> |
− | <li> | + | <li>Remove and dispense 600 µl of supernatant </li> |
− | </li> | + | <li>Re-suspend cells by light vortexing</li> |
− | <li> | + | <li>Plate resuspended cells as above</li> |
− | </li> | + | <li>Incubate overnight at 37°C with plates upside down.</li> |
− | <li>Re-suspend cells by light vortexing | + | |
− | <li> | + | |
− | <li> | + | |
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | </li> | + | |
</ol> | </ol> | ||
+ | <a href=" https://www.neb.com/protocols/2012/05/21/transformation-protocol">Click here for the manufacturer's maual</a> | ||
− | </ | + | </div> |
</div> | </div> | ||
− | |||
</div> | </div> | ||
− | |||
<div class="some-padding"></div> | <div class="some-padding"></div> | ||
<div class="panel-group" id="accordion" role="tablist" aria-multiselectable="true"> | <div class="panel-group" id="accordion" role="tablist" aria-multiselectable="true"> | ||
<div class="panel panel-default"> | <div class="panel panel-default"> | ||
− | <div class="panel-heading" role="tab" id=" | + | <div class="panel-heading" role="tab" id="P8"> |
<h4 class="panel-title"> | <h4 class="panel-title"> | ||
− | <a role="button" data-toggle="collapse" data-parent="#accordion" href="# | + | <a role="button" data-toggle="collapse" data-parent="#accordion" href="#P8-collapse" aria-expanded="false" aria-controls="P8-collapse"> |
<div> | <div> | ||
<div class="col-md-11">Miniprep </div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div> | <div class="col-md-11">Miniprep </div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div> | ||
Line 327: | Line 437: | ||
</div> | </div> | ||
− | <div id=" | + | <div id="P8-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="P8"> |
<div class="panel-body"> | <div class="panel-body"> | ||
− | |||
<ol> | <ol> | ||
<li>Harvest 3 mL cells at 1OD. <br></li> | <li>Harvest 3 mL cells at 1OD. <br></li> | ||
<li>Centrifuge at 14,000 rpm for 30 s. </li> | <li>Centrifuge at 14,000 rpm for 30 s. </li> | ||
− | <li>Discard the supernatant and resuspend the pellet with remnant. | + | <li>Discard the supernatant and resuspend the pellet with remnant.</li> |
− | </li> | + | |
<li>Resuspend with 250 μl Resuspension Buffer.</li> | <li>Resuspend with 250 μl Resuspension Buffer.</li> | ||
<li>Add 250 μl Lysis Buffer. </li> | <li>Add 250 μl Lysis Buffer. </li> | ||
− | <li>Add 350 μl Neutralization Buffer. | + | <li>Add 350 μl Neutralization Buffer.</li> |
− | </li> | + | <li>Centrifuge at 13,000 rpm for 10 mins.</li> |
− | <li>Centrifuge at 13,000 rpm for 10 mins. | + | <li>Transfer supernatent to column.</li> |
− | </li> | + | |
− | <li>Transfer supernatent to column. | + | |
− | + | ||
<li>Centrifuge at 13,000 rpm for 1 min.</li> | <li>Centrifuge at 13,000 rpm for 1 min.</li> | ||
− | <li>Add 700 μl Washing Buffer B. | + | <li>Add 700 μl Washing Buffer B.</li> |
− | </li> | + | |
<li>Centrifuge at 13,000 rpm for 1 min.</li> | <li>Centrifuge at 13,000 rpm for 1 min.</li> | ||
<li>Column dring with centrifuge 13.000rpm for 1 min.</li> | <li>Column dring with centrifuge 13.000rpm for 1 min.</li> | ||
Line 351: | Line 455: | ||
<li>Centrifuge at 13,000 rpm for 1 min.</li> | <li>Centrifuge at 13,000 rpm for 1 min.</li> | ||
</ol> | </ol> | ||
− | + | <a href=" http://eng.intronbio.com/PROTO-PDF/DNA-spin.pdf">Click here for the manufacturer's maual</a> | |
</p> | </p> | ||
</div> | </div> | ||
</div> | </div> | ||
</div> | </div> | ||
− | |||
<div class="some-padding"></div> | <div class="some-padding"></div> | ||
<div class="panel-group" id="accordion" role="tablist" aria-multiselectable="true"> | <div class="panel-group" id="accordion" role="tablist" aria-multiselectable="true"> | ||
<div class="panel panel-default"> | <div class="panel panel-default"> | ||
− | <div class="panel-heading" role="tab" id=" | + | <div class="panel-heading" role="tab" id="P13"> |
<h4 class="panel-title"> | <h4 class="panel-title"> | ||
− | <a role="button" data-toggle="collapse" data-parent="#accordion" href="# | + | <a role="button" data-toggle="collapse" data-parent="#accordion" href="#P13-collapse" aria-expanded="false" aria-controls="P13-collapse"> |
<div> | <div> | ||
− | <div class="col-md-11"> | + | <div class="col-md-11">Culturing medium recipe </div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div> |
</div> | </div> | ||
</a> | </a> | ||
Line 370: | Line 473: | ||
</div> | </div> | ||
− | <div id=" | + | <div id="P13-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="P13"> |
<div class="panel-body"> | <div class="panel-body"> | ||
− | + | LB Broth | |
− | <li> | + | Formulation per one liter |
+ | <ul> | ||
+ | <li>10 g SELECT Peptone 140</li> | ||
+ | <li>5 g SELECT Yeast Extract</li> | ||
+ | <li>5 g Sodium Chloride</li> | ||
+ | </ul> | ||
+ | LB Agar | ||
+ | Formulation per one liter | ||
+ | <ul> | ||
+ | <li>10 g SELECT Peptone 140</li> | ||
+ | <li>5 g SELECT Yeast Extract</li> | ||
+ | <li>5 g Sodium Chloride</li> | ||
+ | <li>12 g SELECT Agar</li> | ||
+ | </ul> | ||
+ | 2XYT | ||
+ | Formulation per one liter | ||
+ | <ul> | ||
+ | <li>16.0 gTryptone </li> | ||
+ | <li>10 g Yeast Extract</li> | ||
+ | <li>5 g Sodium Chloride</li> | ||
+ | Final pH 6.8 ± 0.2 at 25°C | ||
+ | </ul> | ||
+ | </ol> | ||
+ | <a href=" https://www.thermofisher.com/hk/en/home/life-science/cell-culture/microbiological-culture/bacterial-growth-media/lb-broth-and-lb-agar.html">Click here for the manufacturer's maual</a> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
+ | <div class="some-padding"></div> | ||
+ | <div class="some-padding"></div> | ||
+ | <div class="panel-group" id="accordion" role="tablist" aria-multiselectable="true"> | ||
+ | <div class="panel panel-default"> | ||
+ | <div class="panel-heading" role="tab" id="P6"> | ||
+ | <h4 class="panel-title"> | ||
+ | <a role="button" data-toggle="collapse" data-parent="#accordion" href="#P6-collapse" aria-expanded="false" aria-controls="P6-collapse"> | ||
+ | <div> | ||
+ | <div class="col-md-11">Promega S30 Cell Free System </div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div> | ||
+ | </div> | ||
+ | </a> | ||
+ | </h4> | ||
+ | |||
+ | </div> | ||
+ | <div id="P6-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="P6"> | ||
+ | <div class="panel-body"> | ||
+ | <ol> | ||
+ | <li>Set up the following reactions:</li> | ||
<table> | <table> | ||
<tr> | <tr> | ||
− | <th> | + | <th>Component</th> |
<th>Volume</th> | <th>Volume</th> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td> | + | <td>DNA template</td> |
− | <td> | + | <td>≤2 μg </td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td> | + | <td>Amino a cid Mixture (-Met) </td> |
− | <td> | + | <td>5 μl </td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td> | + | <td>S30 Pewmix without amino acids </td> |
− | <td> | + | <td>20 μl </td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td> | + | <td>[35S]Methionine </td> |
− | <td> | + | <td>1 μl</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td> | + | <td>Reverse Primer </td> |
− | <td> | + | <td>2.5 μl</td> |
+ | </tr> | ||
+ | <tr> | ||
+ | <td>S30 Extract, Circular </td> | ||
+ | <td>15 μl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Nucelase-Free Water to final volume of : </td> | ||
+ | <td>50 μl</td> | ||
</tr> | </tr> | ||
</table> | </table> | ||
− | <li> | + | <li>Vortex gently, then centrifuge in a microcentrifuge for 5 seconds to bring the reaction mixture to the bottom of the tube. </li> |
− | <li> | + | <li>Incubate the reactions at 37°C for 60 minutes. </li> |
− | <li> | + | <li>Stop the reactions by placing the tubes in an ice bath for 5 minutes.</li> |
− | < | + | <li>Analyze the results of the reaction. </li> |
+ | </ol> | ||
+ | <a href=" https://www.promega.com/-/media/files/resources/protocols/technical-manuals/0/s30-t7-high-yield-protein-expression-system-protocol.pdf">Click here for the manufacturer's maual</a> | ||
+ | </p> | ||
+ | </div> | ||
+ | </div> | ||
+ | </div> | ||
− | </ | + | <div class="panel-group" id="accordion" role="tablist" aria-multiselectable="true"> |
− | < | + | <div class="panel panel-default"> |
+ | <div class="panel-heading" role="tab" id="P7"> | ||
+ | <h4 class="panel-title"> | ||
+ | <a role="button" data-toggle="collapse" data-parent="#accordion" href="#P7-collapse" aria-expanded="false" aria-controls="P7-collapse"> | ||
+ | <div> | ||
+ | <div class="col-md-11">Characterisation: Protein Purification</div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div> | ||
+ | </div> | ||
+ | </a> | ||
+ | </h4> | ||
− | + | </div> | |
− | <li> | + | <div id="P7-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="P7"> |
− | </li> | + | <div class="panel-body"> |
− | <li>2. | + | Materials: |
− | </li> | + | <ul> |
− | <li> | + | <li>10X TE Buffer</li> |
− | <li> | + | <li>1M Tris HCl Buffer</li> |
− | <li> | + | <li>Protein lysis buffer (PLB): 50 mM Tris (pH 7.5), 50 mM NaCl, dtt. sterilized by autoclaving</li> |
− | <li> | + | <li>Start buffer: 20 mM Tris-HCl, pH 8.0 (At least 1 pH unit above the pI of substance)</li> |
− | <li> | + | <li>Elution Buffer (20mM Tris-HCl, pH 8.0, 1 M NaCl).</li> |
− | <li> | + | <li>Binding buffer: 4 M ammonium sulfate 132.14 g/mol in TE (Tris- EDTA)</li> <li>Wash buffer: 1.3 M ammonium sulfate in TE</li> <li>Equilibrating buffer: 2 M ammonium sulfate in TE</li> |
− | <li> | + | </ul> |
− | <li> | + | Procedures: |
− | <li> | + | <ol> |
+ | <li>Pick single colony of transformed C41 cells to 5ml LB solution with 1x antibiotics to grow starter. <br></li> | ||
+ | <li>1% Inoculation in two 1L conical flask, each with 250 ml 2XYT solution 1x antibiotics overnight. </li> | ||
+ | <li>Spin down 100ml cells in 50 ml falcon. Discard the supernatant.</li> | ||
+ | <li>Wash cell pellet with 40 ml cool TE buffer.</li> | ||
+ | <li>Re-suspend cells with cold 15 ml Protein Lysis Buffer (PLB). </li> | ||
+ | <li>Sonicate on ice.</li> | ||
+ | <li>Spin at 4°C at 13000 rpm for 5 min</li> | ||
+ | <li>Collect the supernatant and dialysis overnight.</li> | ||
+ | <li>Pipette 900 µl of room temperature SOC or LB media into the mixture.</li> | ||
+ | <li>Purify proteins with ion exchange column using start buffer (20 mM Tris-HCl, pH 8.0)</li> | ||
+ | <li>Apply the sample at 5 ml/min for 5 ml columns by pumping it onto the column.</li> | ||
+ | <li>Trace the proteins by naked eyes.</li> | ||
+ | <li>Elute amajLime and mRFP at 0 µM and 30 µM NaCl respectively.</li> | ||
+ | <li>Purify the proteins by HIC column followed by Ion exchange chromatography. Wash the column with 3 mL of Equilibrium buffer after HIC matrix resuspension. </li> | ||
+ | <li>Remove the female luer for buffer running through the column to the waste collection tube.</li> | ||
+ | <li>Mix 1 volume of protein sample to 1 volume of Binding Buffer (~200 µl).</li> | ||
+ | <li>Transfer all 400 µl of protein/Binding Buffer mix to the column carefully without disturbing the top of the bed containing the settled HIC matrix.</li> | ||
+ | <li>Trace the fluorescent protein by naked eyes or blue light.</li> | ||
+ | <li>Wash the resin with 1 ml Wash buffer when the meniscus reaches the top of the bed and the run-through is collected in the waste collection tube.</li> | ||
+ | <li>Add 1 ml TE buffer to the resin and the run-through without fluorescent proteins is collected in the waste collection tube.</li> | ||
+ | <li>Continue adding TE buffer to run through fluorescent proteins and collect with Eppendorf.</li> | ||
+ | <li>Run SDS-PAGE to determine purity.</li> | ||
+ | <li>Determine protein concentration by refractometer.</li> | ||
</ol> | </ol> | ||
− | |||
</p> | </p> | ||
− | + | </div> | |
− | + | ||
</div> | </div> | ||
</div> | </div> | ||
− | |||
<div class="panel-group" id="accordion" role="tablist" aria-multiselectable="true"> | <div class="panel-group" id="accordion" role="tablist" aria-multiselectable="true"> | ||
<div class="panel panel-default"> | <div class="panel panel-default"> | ||
− | <div class="panel-heading" role="tab" id=" | + | <div class="panel-heading" role="tab" id="P14"> |
<h4 class="panel-title"> | <h4 class="panel-title"> | ||
− | <a role="button" data-toggle="collapse" data-parent="#accordion" href="# | + | <a role="button" data-toggle="collapse" data-parent="#accordion" href="#P14-collapse" aria-expanded="false" aria-controls="P14-collapse"> |
<div> | <div> | ||
− | <div class="col-md-11"> | + | <div class="col-md-11">Charaterization: pH stability</div><div class="col-md-1"><i class="fa fa-arrow-down fa-10" aria-hidden="true"></i></div> |
</div> | </div> | ||
</a> | </a> | ||
Line 443: | Line 632: | ||
</div> | </div> | ||
− | <div id=" | + | <div id="P14-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="P14"> |
<div class="panel-body"> | <div class="panel-body"> | ||
− | |||
<ol> | <ol> | ||
− | <li> | + | <li>Prepare the buffers as follow:</li> |
<table> | <table> | ||
<tr> | <tr> | ||
− | <th> | + | <th>pH</th> |
− | <th> | + | <th>Composition</th> |
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td> | + | <td>2</td> |
− | <td>0. | + | <td>0.2M KCl/HCl</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td> | + | <td>4</td> |
− | <td> | + | <td>3M acetate buffer</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td> | + | <td>6</td> |
− | <td> | + | <td>0.5M MES buffer</td> |
</tr> | </tr> | ||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
− | |||
<tr> | <tr> | ||
− | < | + | <td>7</td> |
− | < | + | <td>1M PIPES buffer</td> |
− | + | ||
− | + | ||
</tr> | </tr> | ||
− | |||
− | |||
<tr> | <tr> | ||
− | <td> | + | <td>8</td> |
− | + | <td>1M HEPES buffer</td> | |
− | <td> | + | |
− | + | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td> | + | <td>10</td> |
− | <td> | + | <td>0.2M NaHCO3/NaOH</td> |
− | + | ||
− | + | ||
</tr> | </tr> | ||
− | + | <tr> | |
− | <td> | + | <td>12</td> |
− | <td> | + | <td>0.2M KCl/NaOH</td> |
− | + | ||
− | + | ||
</tr> | </tr> | ||
− | + | </table> | |
− | + | <li>Diluted into buffers ranging in pH from 2-12 in 96-well plates.</li> | |
− | + | <li>Determine absorbance/ fluorescence by Plate reader.</li> | |
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | <li> | + | |
− | <li> | + | |
− | + | ||
− | + | ||
− | + | ||
− | + | ||
− | + | ||
</ol> | </ol> | ||
+ | </p> | ||
− | |||
</div> | </div> | ||
</div> | </div> | ||
</div> | </div> | ||
− | |||
+ | </body> | ||
− | </ | + | </section> |
+ | |||
</html> | </html> | ||
+ | {{Template:Hong_Kong-CUHK_footer}} |
Latest revision as of 14:44, 30 October 2017