|
|
Line 10: |
Line 10: |
| p{ | | p{ |
| color: black; | | color: black; |
− | font-family: Schoolbell;
| |
| } | | } |
| table { | | table { |
Line 67: |
Line 66: |
| <tr> | | <tr> |
| <td>Phusion DNA Polymerase</td> | | <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>
| |
− |
| |
− | </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="P1">
| |
− | <h4 class="panel-title">
| |
− | <a role="button" data-toggle="collapse" data-parent="#accordion" href="#P1-collapse" aria-expanded="false" aria-controls="P1-collapse">
| |
− | <div>
| |
− | <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>
| |
− | </a>
| |
− | </h4>
| |
− |
| |
− | </div>
| |
− | <div id="P1-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="P1">
| |
− | <div class="panel-body">
| |
− |
| |
− | <ol>
| |
− | <li>Prepare your 1% gel by using 0.5g of agarose in 50 ml of TAE buffer.</li>
| |
− | <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>Load your samples and appropriate marker into your wells</li>
| |
− | <li>Incubate at 37°C and 250 rpm for 60-120 minutes.</li>
| |
− | <li>Apply 130 volts to the chamber for 20 mins. </li>
| |
− | <li>Check your gel using a transilluminator or other UV emitting device.</li>
| |
− | <li>Re-suspend cells by light vortexing</li>
| |
− | <li>Plate resuspended cells as above</li>
| |
− | <li>Incubate overnight at 37°C with plates upside down.</li>
| |
− | </ol>
| |
− |
| |
− | </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>
| |
− |
| |
− | </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="P3">
| |
− | <h4 class="panel-title">
| |
− | <a role="button" data-toggle="collapse" data-parent="#accordion" href="#P3-collapse" aria-expanded="false" aria-controls="P3-collapse">
| |
− | <div>
| |
− | <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>
| |
− | </a>
| |
− | </h4>
| |
− |
| |
− | </div>
| |
− | <div id="P3-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="P3">
| |
− | <div class="panel-body">
| |
− | <ol>
| |
− | <li>Prepare the reaction mixture as follow:</li>
| |
− | <table>
| |
− | <tr>
| |
− | <th>Reaction Components</th>
| |
− | <th>Volume</th>
| |
− | </tr>
| |
− | <tr>
| |
− | <td>DNA </td>
| |
− | <td>0.2M KCl/HCl</td>
| |
− | </tr>
| |
− | <tr>
| |
− | <td>DNA template</td>
| |
− | <td>10-200 ng</td>
| |
− | </tr>
| |
− | <tr>
| |
− | <td>10XT4 DNA Ligase Reaction Buffer</td>
| |
− | <td>2 μl</td>
| |
− | </tr>
| |
− | <tr>
| |
− | <td>T4 DNA Ligase</td>
| |
− | <td>1 μl</td>
| |
− | </tr>
| |
− | <tr>
| |
− | <td>Water</td>
| |
− | <td>Top up to 20 μl</td>
| |
− | </tr>
| |
− | </table>
| |
− | <li>Allow the ligation to take place a 22-25°C for 10 minutes.</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>
| |
− | </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="P4">
| |
− | <h4 class="panel-title">
| |
− | <a role="button" data-toggle="collapse" data-parent="#accordion" href="#P4-collapse" aria-expanded="false" aria-controls="P4-collapse">
| |
− | <div>
| |
− | <div class="col-md-11">Ligation</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="P4-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="P4">
| |
− | <div class="panel-body">
| |
− | <ol>
| |
− | <li>Prepare the reagents as follow:</li>
| |
− | <table>
| |
− | <tr>
| |
− | <th>Reaction Components</th>
| |
− | <th>Volume</th>
| |
− | </tr>
| |
− | <tr>
| |
− | <td>DNA template</td>
| |
− | <td>0.5 μg</td>
| |
− | </tr>
| |
− | <tr>
| |
− | <td>10X Buffer</td>
| |
− | <td>2.5 μl</td>
| |
− | </tr>
| |
− | <tr>
| |
− | <td>Enzyme</td>
| |
− | <td>2 μl</td>
| |
− | </tr>
| |
− | <tr>
| |
− | <td>Nuclease-Free Water to final colume of:</td>
| |
− | <td> 25 μl</td>
| |
− | </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>
| |
− | </ol>
| |
− | </p>
| |
− |
| |
− | </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="P9">
| |
− | <h4 class="panel-title">
| |
− | <a role="button" data-toggle="collapse" data-parent="#accordion" href="#P9-collapse" aria-expanded="false" aria-controls="P9-collapse">
| |
− | <div>
| |
− | <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>
| |
− | </a>
| |
− | </h4>
| |
− |
| |
− | </div>
| |
− | <div id="P9-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="P9">
| |
− | <div class="panel-body">
| |
− | <ol>
| |
− | <li>Prepare the Ca/glycerol buffer as follow and flow through 0.22 μM filter.</li>
| |
− | <table>
| |
− | <tr>
| |
− | <th>Composition</th>
| |
− | <th>Volume</th>
| |
− | </tr>
| |
− | <tr>
| |
− | <td>0.6 M CaCl2 </td>
| |
− | <td>10 ml </td>
| |
− | </tr>
| |
− | <tr>
| |
− | <td>0.5 M PIPES pH7.0 </td>
| |
− | <td>2 ml </td>
| |
− | </tr>
| |
− | <tr>
| |
− | <td>Glycerol </td>
| |
− | <td>15 ml </td>
| |
− | </tr>
| |
− | <tr>
| |
− | <td>H2O </td>
| |
− | <td>73 ml </td>
| |
− | </tr>
| |
− | <tr>
| |
− | <td>Total </td>
| |
− | <td>100 ml </td>
| |
− | </tr>
| |
− | </table>
| |
− | <li>Spread the bacterial cell (DH5α) for plasmid replication on the LB. <br></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>
| |
− |
| |
− | </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="P5">
| |
− | <h4 class="panel-title">
| |
− | <a role="button" data-toggle="collapse" data-parent="#accordion" href="#P5-collapse" aria-expanded="false" aria-controls="P5-collapse">
| |
− | <div>
| |
− | <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>
| |
− | </a>
| |
− | </h4>
| |
− |
| |
− | </div>
| |
− | <div id="P5-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="P5">
| |
− | <div class="panel-body">
| |
− |
| |
− | <ol>
| |
− | <li>Thaw 100µL competent E. coli cells on ice for 10 minutes<br></li>
| |
− | <li>Add:</li>
| |
− | <ul>
| |
− | <li>10-20 µl DNA from a ligation reaction mix OR</li>
| |
− | <li>50-100ng DNA of a known plasmid </li>
| |
− | </ul>
| |
− | </li>
| |
− | <li>Place the mixture on ice for 15 minutes.
| |
− | <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>Incubate at 37°C and 250 rpm for 60-120 minutes.</li>
| |
− | <li>Pellet cells at 13000rpm for 1 minutes</li>
| |
− | <li>Remove and dispense 600 µl of supernatant </li>
| |
− | <li>Re-suspend cells by light vortexing</li>
| |
− | <li>Plate resuspended cells as above</li>
| |
− | <li>Incubate overnight at 37°C with plates upside down.</li>
| |
− | </ol>
| |
− |
| |
− | </div>
| |
− | </div>
| |
− | </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="P8">
| |
− | <h4 class="panel-title">
| |
− | <a role="button" data-toggle="collapse" data-parent="#accordion" href="#P8-collapse" aria-expanded="false" aria-controls="P8-collapse">
| |
− | <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>
| |
− | </a>
| |
− | </h4>
| |
− |
| |
− | </div>
| |
− | <div id="P8-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="P8">
| |
− | <div class="panel-body">
| |
− | <ol>
| |
− | <li>Harvest 3 mL cells at 1OD. <br></li>
| |
− | <li>Centrifuge at 14,000 rpm for 30 s. </li>
| |
− | <li>Discard the supernatant and resuspend the pellet with remnant.</li>
| |
− | <li>Resuspend with 250 μl Resuspension Buffer.</li>
| |
− | <li>Add 250 μl Lysis Buffer. </li>
| |
− | <li>Add 350 μl Neutralization Buffer.</li>
| |
− | <li>Centrifuge at 13,000 rpm for 10 mins.</li>
| |
− | <li>Transfer supernatent to column.</li>
| |
− | <li>Centrifuge at 13,000 rpm for 1 min.</li>
| |
− | <li>Add 700 μl Washing Buffer B.</li>
| |
− | <li>Centrifuge at 13,000 rpm for 1 min.</li>
| |
− | <li>Column dring with centrifuge 13.000rpm for 1 min.</li>
| |
− | <li>Inoculate with 30 μl distilled water. </li>
| |
− | <li>Centrifuge at 13,000 rpm for 1 min.</li>
| |
− | </ol>
| |
− |
| |
− | </p>
| |
− | </div>
| |
− | </div>
| |
− | </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="P13">
| |
− | <h4 class="panel-title">
| |
− | <a role="button" data-toggle="collapse" data-parent="#accordion" href="#P13-collapse" aria-expanded="false" aria-controls="P13-collapse">
| |
− | <div>
| |
− | <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>
| |
− | </a>
| |
− | </h4>
| |
− |
| |
− | </div>
| |
− | <div id="P13-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="P13">
| |
− | <div class="panel-body">
| |
− | LB Broth
| |
− | 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>
| |
− |
| |
− | </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">
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− | <div class="panel-body">
| |
− | <ol>
| |
− | <li>Set up the following reactions:</li>
| |
− | <table>
| |
− | <tr>
| |
− | <th>Component</th>
| |
− | <th>Volume</th>
| |
− | </tr>
| |
− | <tr>
| |
− | <td>DNA template</td>
| |
− | <td>≤2 μg </td>
| |
− | </tr>
| |
− | <tr>
| |
− | <td>Amino a cid Mixture (-Met) </td>
| |
− | <td>5 μl </td>
| |
− | </tr>
| |
− | <tr>
| |
− | <td>S30 Pewmix without amino acids </td>
| |
− | <td>20 μl </td>
| |
− | </tr>
| |
− | <tr>
| |
− | <td>[35S]Methionine </td>
| |
− | <td>1 μl</td>
| |
− | </tr>
| |
− | <tr>
| |
− | <td>Reverse Primer </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>
| |
− | </table>
| |
− | <li>Vortex gently, then centrifuge in a microcentrifuge for 5 seconds to bring the reaction mixture to the bottom of the tube. </li>
| |
− | <li>Incubate the reactions at 37°C for 60 minutes. </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>
| |
− |
| |
− | </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>
| |
− | <div id="P7-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="P7">
| |
− | <div class="panel-body">
| |
− | Materials:
| |
− | <ul>
| |
− | <li>10X TE Buffer</li>
| |
− | <li>1M Tris HCl Buffer</li>
| |
− | <li>Protein lysis buffer (PLB): 50 mM Tris (pH 7.5), 50 mM NaCl, dtt. sterilized by autoclaving</li>
| |
− | <li>Start buffer: 20 mM Tris-HCl, pH 8.0 (At least 1 pH unit above the pI of substance)</li>
| |
− | <li>Elution Buffer (20mM Tris-HCl, pH 8.0, 1 M NaCl).</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>
| |
− | </ul>
| |
− | Procedures:
| |
− | <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>
| |
− | </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="P14">
| |
− | <h4 class="panel-title">
| |
− | <a role="button" data-toggle="collapse" data-parent="#accordion" href="#P14-collapse" aria-expanded="false" aria-controls="P14-collapse">
| |
− | <div>
| |
− | <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>
| |
− | </a>
| |
− | </h4>
| |
− |
| |
− | </div>
| |
− | <div id="P14-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="P14">
| |
− | <div class="panel-body">
| |
− | <ol>
| |
− | <li>Prepare the buffers as follow:</li>
| |
− | <table>
| |
− | <tr>
| |
− | <th>pH</th>
| |
− | <th>Composition</th>
| |
− | </tr>
| |
− | <tr>
| |
− | <td>2</td>
| |
− | <td>0.2M KCl/HCl</td>
| |
− | </tr>
| |
− | <tr>
| |
− | <td>4</td>
| |
− | <td>3M acetate buffer</td>
| |
− | </tr>
| |
− | <tr>
| |
− | <td>6</td>
| |
− | <td>0.5M MES buffer</td>
| |
− | </tr>
| |
− | <tr>
| |
− | <td>7</td>
| |
− | <td>1M PIPES buffer</td>
| |
− | </tr>
| |
− | <tr>
| |
− | <td>8</td>
| |
− | <td>1M HEPES buffer</td>
| |
− | </tr>
| |
− | <tr>
| |
− | <td>10</td>
| |
− | <td>0.2M NaHCO3/NaOH</td>
| |
− | </tr>
| |
− | <tr>
| |
− | <td>12</td>
| |
− | <td>0.2M KCl/NaOH</td>
| |
− | </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>
| |
− | </ol>
| |
− | </p>
| |
− |
| |
− | </div>
| |
− | </div>
| |
− | </div>
| |
− |
| |
− |
| |
− | </section>
| |
− |
| |
− |
| |
− |
| |
− | </body>
| |
− | </html>
| |