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+ | <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="P6"> |
<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="#P6-collapse" aria-expanded="false" aria-controls="P6-collapse"> |
<div> | <div> | ||
− | <div class="col-md-11"> | + | <div class="col-md-11">Charaterization </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 id=" | + | <div id="P6-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="P6"> |
<div class="panel-body"> | <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> | <ol> | ||
− | <li> | + | <li>Pick single colony of transformed C41 cells to 5ml LB solution with 1x antibiotics to grow starter. <br></li> |
− | <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> | + | <li>Wash cell pellet with 40 ml cool TE buffer.</li> |
− | <li> | + | <li>Re-suspend cells with cold 15 ml Protein Lysis Buffer (PLB). </li> |
− | < | + | <li>Sonicate on ice.</li> |
− | </li> | + | <li>Spin at 4°C at 13000 rpm for 5 min</li> |
− | <li> | + | <li>Collect the supernatant and dialysis overnight.</li> |
− | <li> | + | |
− | <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>Purify proteins with ion exchange column using start buffer (20 mM Tris-HCl, pH 8.0)</li> |
− | <li> | + | <li>Apply the sample at 5 ml/min for 5 ml columns by pumping it onto the column.</li> |
− | <li> | + | <li>Trace the proteins by naked eyes.</li> |
− | <li> | + | <li>Elute amajLime and mRFP at 0 µM and 30 µM NaCl respectively.</li> |
− | <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> | + | <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> | |
− | + | ||
</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"> | ||
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<tr> | <tr> | ||
<td>7</td> | <td>7</td> | ||
− | <td>1M PIPES buffer/td> | + | <td>1M PIPES buffer</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
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</tr> | </tr> | ||
</table> | </table> | ||
− | <li>Diluted into buffers ranging in pH from | + | <li>Diluted into buffers ranging in pH from 2-12 in 96-well plates.</li> |
<li>Determine absorbance/ fluorescence by Plate reader.</li> | <li>Determine absorbance/ fluorescence by Plate reader.</li> | ||
</ol> | </ol> | ||
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</div> | </div> | ||
</div> | </div> | ||
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<th>Reaction Components</th> | <th>Reaction Components</th> | ||
<th>Volume</th> | <th>Volume</th> | ||
− | |||
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− | |||
− | |||
</tr> | </tr> | ||
<tr> | <tr> | ||
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<tr> | <tr> | ||
<td>Enzyme</td> | <td>Enzyme</td> | ||
− | <td>2 μl/td> | + | <td>2 μl</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
− | <td>Water</td> | + | <td>Nuclease-Free Water to final colume of:</td> |
− | <td> | + | <td> 25 μl</td> |
</tr> | </tr> | ||
</table> | </table> | ||
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</div> | </div> | ||
</div> | </div> | ||
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<div class="some-padding"></div> | <div class="some-padding"></div> | ||
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<li>Pick single colony in 5 ml LB culture medium and grow overnight at 37 °C by shaking at ~220 rpm. </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>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>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>Collect the cell by centrifuging at 1000 g for 10 min at 4°C. </li> | ||
Line 401: | Line 277: | ||
</div> | </div> | ||
</div> | </div> | ||
+ | |||
<div class="some-padding"></div> | <div class="some-padding"></div> | ||
<div class="some-padding"></div> | <div class="some-padding"></div> | ||
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</tr> | </tr> | ||
<li>Set up thermocycler for PCR reaction as follow:</li> | <li>Set up thermocycler for PCR reaction as follow:</li> | ||
− | |||
<tr> | <tr> | ||
<td>Initial denaturation </td> | <td>Initial denaturation </td> | ||
Line 493: | Line 369: | ||
</tr> | </tr> | ||
</table> | </table> | ||
− | |||
<li>Add the other Component in the tube. If the added volume is less, pipetting up and down | <li>Add the other Component in the tube. If the added volume is less, pipetting up and down | ||
is necessary. </li> | is necessary. </li> | ||
Line 505: | Line 380: | ||
</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 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">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="P2-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="P2"> | ||
+ | <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="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="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">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="P5-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="P5"> | ||
+ | <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="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">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="P1-collapse" class="panel-collapse collapse" role="tabpanel" aria-labelledby="P1"> | ||
+ | <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> | ||
Revision as of 04:57, 23 August 2017