Difference between revisions of "Team:NCKU Tainan/Protocol"
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<html> | <html> | ||
| − | + | <head> | |
| − | + | <link rel="stylesheet" href="https://2018.igem.org/Template:NCKU_Tainan/css/protocol_style?action=raw&ctype=text/css"> | |
| − | + | </head> | |
| − | + | <body data-spy="scroll" data-target=".navbar-example"> | |
| − | + | <div class="container content"> | |
| + | <div class="headstyle"> | ||
<h1 class="head">Protocol</h1> | <h1 class="head">Protocol</h1> | ||
| − | <div class="navbar-example"> | + | </div> |
| − | + | <div class="righttitle"> | |
| − | + | <h6 class="subtitle">Read </h6> | |
| − | + | </div> | |
| − | + | <div class="navbar-example"> | |
| − | + | <div class="row"> | |
| − | + | <div class="col-12"> | |
| − | + | <div data-spy="scroll" data-target="#sidelist" data-offset="0" class="scrollspy-example"> | |
| − | + | <div class="container"> | |
| − | + | <div class="row"> | |
| − | + | <div class="card-deck"> | |
| − | + | <div class="card col-md-4"> | |
| − | + | <div class="action-button" type="button" class="btn btn-primary" data-toggle="modal" | |
| − | + | data-target="#Dry_cell_weight"> | |
| − | + | <div class="post"> | |
| − | + | <span class="folded-corner"></span> | |
| + | <img class="card-img-top" src="https://static.igem.org/mediawiki/2018/1/1f/T--NCKU_Tainan--protocol_Dry_cell_weight.jpg" | ||
| + | alt="Dry cell weight"> | ||
| + | </div> | ||
| + | <div class="card-body"> | ||
| + | <h5 class="card-title">Dry Cell Weight</h5> | ||
</div> | </div> | ||
</div> | </div> | ||
| − | + | </div> | |
| − | + | <div class="modal" id="Dry_cell_weight" tabindex="-1" role="dialog"> | |
| − | + | <div class="modal-dialog" role="document"> | |
| − | + | <div class="modal-content"> | |
| − | + | <div class="modal-header">Dry Cell Weight | |
| − | + | <button type="button" class="close" data-dismiss="modal" aria-label="Close"> | |
| − | + | <span aria-hidden="true">×</span> | |
| − | + | </button> | |
| − | + | </div> | |
| − | + | <div class="modal-body"> | |
| − | + | <ol> | |
| − | + | <li class="licontent">Preculture the bacteria overnight in 4ml | |
| − | + | LB with selective antibiotic.</li> | |
| − | + | <li class="licontent">Refresh the precultured bacteria in 20 ml | |
| − | + | of M9 xylose in flask. </li> | |
| − | + | <li class="licontent">Culture the bacteria for 24 hr in normal | |
| − | + | incubator with 177 rpm and 37℃ condition in 24 hr.</li> | |
| − | + | <li class="licontent">Measure the optical density of the sample | |
| − | + | with 600 nm light wavelength, and then diluted the optical | |
| − | + | density value to 0.25 ,0.5, 0.75, 1 in 4ml of bacteria | |
| − | + | respectively.</li> | |
| − | + | <li class="licontent">Measure the weight of an empty eppendorf.</li> | |
| − | + | <li class="licontent">Transfer 4 ml of bacteria to eppendorf, | |
| − | + | and then centrifuge them at 16000xg for 3 min.</li> | |
| + | <li class="licontent">Put the eppendorfs in the 60℃ | ||
| + | high-temperature oven with cap opened for 12 hr.</li> | ||
| + | <li class="licontent">Measure the net weight of the bacteria | ||
| + | after 12 hr.</li> | ||
| + | <li class="licontent">Analyze the dry cell weight and draw the | ||
| + | regression line of it.</li> | ||
| + | </ol> | ||
| + | </div> | ||
| + | <div class="modal-footer"> | ||
| + | <button type="button" class="btn btn-secondary" data-dismiss="modal">Close</button> | ||
</div> | </div> | ||
</div> | </div> | ||
</div> | </div> | ||
| + | </div> | ||
| − | + | <div class="card col-md-4"> | |
| − | + | <div class="action-button" type="button" class="btn btn-primary" data-toggle="modal" | |
| − | + | data-target="#Minimal_Salts_Medium_Xylose_Preparation"> | |
| − | + | <div class="post"> | |
| − | + | <span class="folded-corner"></span> | |
| − | + | <img class="card-img-top" src="https://static.igem.org/mediawiki/2018/4/4f/T--NCKU_Tainan--protocol_M9_Medium_Xylose_Preparation.jpg" | |
| − | + | alt="assay"> | |
| − | + | </div> | |
| − | + | <div class="card-body"> | |
| + | <h5 class="card-title">Minimal Salts (M9) Medium Xylose Preparation</h5> | ||
</div> | </div> | ||
</div> | </div> | ||
| − | + | </div> | |
| − | + | <div class="modal" id="Minimal_Salts_Medium_Xylose_Preparation" tabindex="-1" role="dialog"> | |
| − | + | <div class="modal-dialog" role="document"> | |
| − | + | <div class="modal-content"> | |
| − | + | <div class="modal-header">Minimal Salts (M9) Medium Xylose Preparation | |
| − | + | <button type="button" class="close" data-dismiss="modal" aria-label="Close"> | |
| − | + | <span aria-hidden="true">×</span> | |
| − | + | </button> | |
| − | + | </div> | |
| − | + | <div class="modal-body"> | |
| − | + | <p class="blackp"> | |
| − | + | M9 medium contains essential salts and nitrogen. | |
| − | + | It contains 33.9 g/L Na<sub>2</sub>HPO<sub>4</sub>·7H<sub>2</sub>O, | |
| − | + | 15 g/L KH<sub>2</sub>PO<sub>4</sub>, 5 g/L NH<sub>4</sub>Cl and | |
| − | + | 2.5 g/L NaCl; | |
| − | + | suitable for recombinant <i>E. coli</i> strains | |
| − | + | </p> | |
| − | + | <ul> | |
| − | + | <li class="licontent">Minimal salts (M9) medium is suitable for | |
| − | + | non-selective cultivation of <i>E. coli</i> strains for | |
| − | + | cloning, | |
| − | + | production of DNA, plasmid DNA and recombinant proteins. | |
| − | + | </li> | |
| − | + | <li class="licontent">Suitable for selective cultivation when | |
| − | + | appropriate antibiotics are added.</li> | |
| − | + | </ul> | |
| − | + | <table class="centertable"> | |
| − | + | <tr> | |
| − | + | <th>Substances</th> | |
| − | + | <th></th> | |
| − | + | <th>Volume in ml</th> | |
| − | + | <th>Volume(M)</th> | |
| − | + | </tr> | |
| − | + | <tr> | |
| − | + | <td colspan="2">M9 salt solution (10X)</td> | |
| − | + | <td></td> | |
| − | + | <td></td> | |
| − | + | </tr> | |
| − | + | <tr> | |
| − | + | <td></td> | |
| − | + | <td>Na<sub>2</sub>HPO<sub>4</sub></td> | |
| − | + | <td rowspan="4">100</td> | |
| − | + | <td>33.7 mM</td> | |
| − | + | </tr> | |
| − | + | <tr> | |
| − | + | <td></td> | |
| − | + | <td>KH<sub>2</sub>PO<sub>4</sub></td> | |
| − | + | <td>22.0 mM</td> | |
| − | + | </tr> | |
| − | + | <tr> | |
| − | + | <td></td> | |
| − | + | <td>NH<sub>4</sub>Cl</td> | |
| − | + | <td>9.35 mM</td> | |
| − | + | </tr> | |
| − | + | <tr> | |
| − | + | <td></td> | |
| − | + | <td>NaCl</td> | |
| − | + | <td>8.55 mM</td> | |
| − | + | </tr> | |
| − | + | <tr> | |
| − | + | <td>20% xylose</td> | |
| − | + | <td></td> | |
| − | + | <td>20</td> | |
| − | + | <td>0.4 %</td> | |
| − | + | </tr> | |
| − | + | <tr> | |
| − | + | <td>1 M MgSO<sub>4</sub></td> | |
| − | + | <td></td> | |
| − | + | <td>1</td> | |
| − | + | <td>1 mM</td> | |
| − | + | </tr> | |
| − | + | <tr> | |
| − | + | <td>1 M CaCl<sub>2</sub></td> | |
| + | <td></td> | ||
| + | <td>0.3</td> | ||
| + | <td>0.3 mM</td> | ||
| + | </tr> | ||
| + | <!--tr> | ||
<td>biotin (1 mg/ml)</td> | <td>biotin (1 mg/ml)</td> | ||
<td></td> | <td></td> | ||
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<td>1X</td> | <td>1X</td> | ||
</tr--> | </tr--> | ||
| − | + | </table> | |
| − | + | <ol> | |
| − | + | <li class="licontent">M9 salt solution (10X)</li> | |
| − | + | <p class="blackp">Na<sub>2</sub>HPO<sub>4</sub>·2H<sub>2</sub>O | |
| − | + | 75.2 g/L</p> | |
| − | + | <p class="blackp">KH<sub>2</sub>PO<sub>4</sub> 30 g/L</p> | |
| − | + | <p class="blackp">NaCl 5 g/L</p> | |
| − | + | <p class="blackp">NH<sub>4</sub>Cl 5 g/L</p> | |
| − | + | <p class="blackp">- Dissolve the salts in 800 ml water</p> | |
| − | + | <p class="blackp">- Add water to a final volume of 1 L</p> | |
| + | <p class="blackp">- Autoclave for 15 min at 121°C</p> | ||
| − | + | <li class="licontent">20% Xylose</li> | |
| − | + | <p class="blackp">- Add 100 g xylose to 440 ml water</p> | |
| − | + | <p class="blackp">- Add water to final volume 500ml</p> | |
| − | + | <p class="blackp">- Autoclave for 15 min at 121°C</p> | |
| − | + | <li class="licontent">1 M MgSO<sub>4</sub></li> | |
| − | + | <p class="blackp">- Dissolve 24.65 g MgSO<sub>4</sub>-7H<sub>2</sub>O | |
| − | + | in 87 ml water</p> | |
| − | + | <p class="blackp">- Add water to final volume 100ml</p> | |
| + | <p class="blackp">- Autoclave for 15 min at 121°C</p> | ||
| − | + | <li class="licontent">1 M CaCl<sub>2</sub></li> | |
| − | + | <p class="blackp">- Dissolve 14.70 g CaCl<sub>2</sub>-2H<sub>2</sub>O | |
| − | + | in 94.5 ml water</p> | |
| + | <p class="blackp">- Autoclave for 15 min at 121°C</p> | ||
| − | + | <!--li class="licontent">Biotin (1 mg/ml)</li> | |
<p class="blackp">- Dissolve 50 mg biotin in 45 ml water</p> | <p class="blackp">- Dissolve 50 mg biotin in 45 ml water</p> | ||
<p class="blackp">- Add small aliquots of 1N NaOH until the biotin has dissolved</p> | <p class="blackp">- Add small aliquots of 1N NaOH until the biotin has dissolved</p> | ||
| Line 198: | Line 224: | ||
<p class="blackp">498 mg FeCl<sub>3</sub> (anhydrous)</p> | <p class="blackp">498 mg FeCl<sub>3</sub> (anhydrous)</p> | ||
<p class="blackp">84 mg ZnCl<sub>2</sub></p> | <p class="blackp">84 mg ZnCl<sub>2</sub></p> | ||
| − | <p class="blackp">765 µl 0.1 M | + | <p class="blackp">765 µl 0.1 M CuCl<sub>2</sub>-2H<sub>2</sub>O 1.70 g/100 ml</p> |
| − | <p class="blackp">210 µl 0.2 M | + | <p class="blackp">210 µl 0.2 M CoCl<sub>2</sub>-6H<sub>2</sub>O 4.76 g/100 ml</p> |
| − | <p class="blackp">1.6 ml 0.1 M | + | <p class="blackp">1.6 ml 0.1 M H<sub>3</sub>BO<sub>3</sub> 0.62 g/100 ml</p> |
| − | <p class="blackp">8.1 µl 1 M | + | <p class="blackp">8.1 µl 1 M MnCl<sub>2</sub>-4H<sub>2</sub>O 19.8 g/100 ml</p--> |
| − | + | </ol> | |
| − | + | </div> | |
| − | + | <div class="modal-footer"> | |
| − | + | <button type="button" class="btn btn-secondary" data-dismiss="modal">Close</button> | |
| − | + | ||
</div> | </div> | ||
</div> | </div> | ||
</div> | </div> | ||
| + | </div> | ||
| − | + | <div class="card col-md-4"> | |
| − | + | <div class="action-button" type="button" class="btn btn-primary" data-toggle="modal" | |
| − | + | data-target="#PRK_Toxicity_Test"> | |
| − | + | <div class="post"> | |
| − | + | <span class="folded-corner"></span> | |
| − | + | <img class="card-img-top" src="https://static.igem.org/mediawiki/2018/d/d9/T--NCKU_Tainan--protocol_PRK_Toxicity_Test.jpg" | |
| − | + | alt="Total solution"> | |
| − | + | </div> | |
| − | + | <div class="card-body"> | |
| + | <h5 class="card-title">PRK Toxicity Test</h5> | ||
</div> | </div> | ||
</div> | </div> | ||
| − | + | </div> | |
| − | + | <div class="modal" id="PRK_Toxicity_Test" tabindex="-1" role="dialog"> | |
| − | + | <div class="modal-dialog" role="document"> | |
| − | + | <div class="modal-content"> | |
| − | + | <div class="modal-header">PRK Toxicity Test | |
| − | + | <button type="button" class="close" data-dismiss="modal" aria-label="Close"> | |
| − | + | <span aria-hidden="true">×</span> | |
| − | + | </button> | |
| − | + | </div> | |
| − | + | <div class="modal-body"> | |
| − | + | <ol> | |
| − | + | <li class="licontent">Preculture the bacteria overnight in 4ml | |
| − | + | LB with antibiotic.</li> | |
| − | + | <li class="licontent">Refresh the precultured bacteria in 4ml | |
| − | + | of M9 xylose in test tube.</li> | |
| − | + | <li class="licontent">Culture in the normal incubator for 12 hr | |
| − | + | with 177 rpm</li> | |
| − | + | <li class="licontent">Measure the optical density of the sample | |
| − | + | with 600 nm light wavelength at the beginning (0 hr) , and | |
| + | also measure them after 12 hr. All samples should be | ||
| + | measured in triplicate.</li> | ||
| + | </ol> | ||
| + | </div> | ||
| + | <div class="modal-footer"> | ||
| + | <button type="button" class="btn btn-secondary" data-dismiss="modal">Close</button> | ||
</div> | </div> | ||
</div> | </div> | ||
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</div> | </div> | ||
</div> | </div> | ||
| + | </div> | ||
| − | + | <div class="row"> | |
| − | + | <div class="card-deck"> | |
| − | + | <div class="card col-md-4"> | |
| − | + | <div class="action-button" type="button" class="btn btn-primary" data-toggle="modal" | |
| − | + | data-target="#DNS_Reductive_Sugar_Measurement"> | |
| − | + | <div class="post"> | |
| − | + | <span class="folded-corner"></span> | |
| − | + | <img class="card-img-top" src="https://static.igem.org/mediawiki/2018/9/99/T--NCKU_Tainan--protocol_DNS.jpg" | |
| − | + | alt="DNS"> | |
| − | + | </div> | |
| − | + | <div class="card-body"> | |
| + | <h5 class="card-title">DNS Reductive Sugar Measurement</h5> | ||
</div> | </div> | ||
</div> | </div> | ||
| − | + | </div> | |
| − | + | <div class="modal" id="DNS_Reductive_Sugar_Measurement" tabindex="-1" role="dialog"> | |
| − | + | <div class="modal-dialog" role="document"> | |
| − | + | <div class="modal-content"> | |
| − | + | <div class="modal-header">DNS Reductive Sugar Measurement | |
| − | + | <button type="button" class="close" data-dismiss="modal" aria-label="Close"> | |
| − | + | <span aria-hidden="true">×</span> | |
| − | </ | + | </button> |
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</div> | </div> | ||
| − | + | <div class="modal-body"> | |
| − | + | <h3>DNS solution preparation:</h3> | |
| + | <ol> | ||
| + | <li class="licontent">Disolve 2.5g of 3,5-Dinitrosalicylic acid | ||
| + | (DNS) to 150ml double distilled water.</li> | ||
| + | <li class="licontent">Heat to solution to 45 degree Celsius and | ||
| + | add 4g of NaOH. Stir the solution until it is transparent.</li> | ||
| + | <li class="licontent">Add 75g of potassium sodium tartrate and | ||
| + | add water to 250 ml.</li> | ||
| + | <li class="licontent">Keep the solution without light exposure. | ||
| + | The solution can be used after 7 days.</li> | ||
| + | </ol> | ||
| − | + | <h3>Principle:</h3> | |
| − | + | <p class="blackp"> | |
| − | + | Under base solution, DNS will turn to brown color while | |
| − | + | reacting with reductive sugar in high temperature. In the | |
| − | <img class=" | + | specific temperature |
| + | range, the color will have linear relationship with the | ||
| + | reductive sugar concentration. | ||
| + | </p> | ||
| + | |||
| + | <h3>Calibration:</h3> | ||
| + | <ol> | ||
| + | <li class="licontent">Prepare glucose or xylose water solution | ||
| + | to the following | ||
| + | concentration: 0, 0.2, 0.4, 0.8, 1.0, 1.2, 1.6, 2 g/L.</li> | ||
| + | <li class="licontent">Take 200 ul of sample, add 200 ul of DNS | ||
| + | solution.</li> | ||
| + | <li class="licontent">Heat the sample at 100 degree Celsius for | ||
| + | 10mins.</li> | ||
| + | <li class="licontent">Cool down the sample with ice to room | ||
| + | temperature.</li> | ||
| + | <li class="licontent">Measure the optical density of the sample | ||
| + | with 540nm light wavelength.</li> | ||
| + | <li class="licontent">Draw the graph of sugar concentration | ||
| + | with respect to optical density.</li> | ||
| + | </ol> | ||
| + | |||
| + | <h3>Calibration results:</h3> | ||
| + | <img class="contentimg" src="https://static.igem.org/mediawiki/2018/f/fa/T--NCKU_Tainan--home_42846829_332575510640801_8298239152197992448_n.png"> | ||
| + | <h3>Measurement:</h3> | ||
| + | <ol> | ||
| + | <li class="licontent">Take 200 ul of sample, add 200 ul of DNS | ||
| + | solution.</li> | ||
| + | <li class="licontent">Heat the sample at 100 degree Celsius for | ||
| + | 10mins.</li> | ||
| + | <li class="licontent">Cool down the sample with ice to room | ||
| + | temperature.</li> | ||
| + | <li class="licontent">Measure the optical density of the sample | ||
| + | with 540nm light wavelength.</li> | ||
| + | <li class="licontent">Get the concentration of reductive sugar | ||
| + | from the Calibration graph.</li> | ||
| + | </ol> | ||
</div> | </div> | ||
| − | <div class=" | + | <div class="modal-footer"> |
| − | < | + | <button type="button" class="btn btn-secondary" data-dismiss="modal">Close</button> |
</div> | </div> | ||
</div> | </div> | ||
</div> | </div> | ||
| − | + | </div> | |
| − | + | ||
| − | + | <div class="card col-md-4"> | |
| − | + | <div class="action-button" type="button" class="btn btn-primary" data-toggle="modal" | |
| − | + | data-target="#Short_term"> | |
| − | + | <div class="post"> | |
| − | + | <span class="folded-corner"></span> | |
| − | + | <img class="card-img-top" src="https://static.igem.org/mediawiki/2018/1/10/T--NCKU_Tainan--protocol_short_term.jpg" | |
| − | + | alt="Short term"> | |
| − | + | </div> | |
| − | + | <div class="card-body"> | |
| − | + | <h5 class="card-title">Short Term pH Sensing Systen Measurement</h5> | |
| − | + | ||
| − | + | ||
| − | + | ||
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</div> | </div> | ||
</div> | </div> | ||
| − | + | </div> | |
| − | + | <div class="modal" id="Short_term" tabindex="-1" role="dialog"> | |
| − | + | <div class="modal-dialog" role="document"> | |
| − | + | <div class="modal-content"> | |
| − | + | <div class="modal-header">Short Term pH Sensing Systen Measurement | |
| − | < | + | <button type="button" class="close" data-dismiss="modal" aria-label="Close"> |
| + | <span aria-hidden="true">×</span> | ||
| + | </button> | ||
</div> | </div> | ||
| − | <div class=" | + | <div class="modal-body"> |
| − | < | + | <ol> |
| + | <li class="licontent">Preculture the bacteria o/n in LB, and | ||
| + | prepared 8 different | ||
| + | (pH4, 4.25, 4.5, 4.75, 5, 5.5, 6,7) pH value of M9 buffer.</li> | ||
| + | <li class="licontent">Culture the bacteria in 6 ml LB with | ||
| + | selective antibiotic to O.D. 0.6 (about 1.5-2hr)</li></br> | ||
| + | <li class="licontent">Divide 6 ml of bacteria into 8 | ||
| + | eppendorfs, and centrifuged them at 1300 rpm | ||
| + | for 1 mins in 37 ℃, then discard the supernatant | ||
| + | completely.</li> | ||
| + | <li class="licontent">Add 700 μl per different pH value of M9 | ||
| + | buffer into 8 different eppendorfs, | ||
| + | respectively . And resuspend the cells completely by | ||
| + | pipetting.</li> | ||
| + | <li class="licontent">Add 200 μl of bacteria in 96 well (This | ||
| + | step need triple repetition.)</li> | ||
| + | <li class="licontent">Measure the O.D.595 nm at the first and | ||
| + | the last time point , and the | ||
| + | fluorescence value (485-535nm ) at every 180 sec, within 30 | ||
| + | mins.</li> | ||
| + | </ol> | ||
</div> | </div> | ||
| − | + | <div class="modal-footer"> | |
| − | + | <button type="button" class="btn btn-secondary" data-dismiss="modal">Close</button> | |
| − | + | ||
| − | + | ||
| − | <div class="modal- | + | |
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</div> | </div> | ||
</div> | </div> | ||
</div> | </div> | ||
</div> | </div> | ||
| − | |||
| − | + | <div class="card col-md-4"> | |
| − | + | <div class="action-button" type="button" class="btn btn-primary" data-toggle="modal" | |
| − | + | data-target="#Long_term_pH_alert_system_measurement"> | |
| − | + | <div class="post"> | |
| − | + | <span class="folded-corner"></span> | |
| − | + | <img class="card-img-top" src="https://static.igem.org/mediawiki/2018/b/b2/T--NCKU_Tainan--protocol_Long_term.jpg" | |
| − | + | alt="Long term"> | |
| − | + | </div> | |
| − | + | <div class="card-body"> | |
| − | + | <h5 class="card-title">Long Term pH Sensing System Measurement</h5> | |
| − | + | ||
</div> | </div> | ||
</div> | </div> | ||
| − | + | </div> | |
| − | + | <div class="modal" id="Long_term_pH_alert_system_measurement" tabindex="-1" role="dialog"> | |
| − | + | <div class="modal-dialog" role="document"> | |
| − | + | <div class="modal-content"> | |
| − | + | <div class="modal-header">Long Term pH Sensing System Measurement | |
| − | + | <button type="button" class="close" data-dismiss="modal" aria-label="Close"> | |
| − | + | <span aria-hidden="true">×</span> | |
| − | + | </button> | |
| − | + | </div> | |
| − | + | <div class="modal-body"> | |
| − | + | <ol> | |
| − | + | <li class="licontent">Preculture the bacteria o/n in LB, and | |
| − | + | prepared 8 different | |
| − | + | (pH4, 5, 6, 7) pH value of M9 buffer.</li> | |
| − | + | <li class="licontent">Add 1/100 of the bacteria in 20 ml of | |
| − | + | different pH value of M9 buffer with | |
| − | + | selective antibiotic.</li> | |
| − | + | <li class="licontent">Culture the bacteria in the incubator in | |
| − | + | 37℃for 24 hr.</li> | |
| − | + | <li class="licontent">Measure the O.D. value (595 nm) and the | |
| + | fluorescence value (485-535nm ) at every 1 hr , within 24 | ||
| + | hr.</li> | ||
| + | </ol> | ||
| + | </div> | ||
| + | <div class="modal-footer"> | ||
| + | <button type="button" class="btn btn-secondary" data-dismiss="modal">Close</button> | ||
</div> | </div> | ||
</div> | </div> | ||
</div> | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| − | + | <div class="row"> | |
| − | + | <div class="card-deck"> | |
| − | + | <div class="card col-md-4"> | |
| − | + | <div class="action-button" type="button" class="btn btn-primary" data-toggle="modal" | |
| − | + | data-target="#Total_Solution"> | |
| − | + | <div class="post"> | |
| − | + | <span class="folded-corner"></span> | |
| − | + | <img class="card-img-top" src="https://static.igem.org/mediawiki/2018/8/84/T--NCKU_Tainan--protocol_total_solution.png" | |
| − | + | alt="Total solution"> | |
| + | </div> | ||
| + | <div class="card-body"> | ||
| + | <h5 class="card-title">Total Solution</h5> | ||
</div> | </div> | ||
</div> | </div> | ||
| − | + | </div> | |
| − | + | <div class="modal" id="Total_Solution" tabindex="-1" role="dialog"> | |
| − | + | <div class="modal-dialog" role="document"> | |
| − | + | <div class="modal-content"> | |
| − | + | <div class="modal-header">Total Solution | |
| − | + | <button type="button" class="close" data-dismiss="modal" aria-label="Close"> | |
| − | + | <span aria-hidden="true">×</span> | |
| − | + | </button> | |
| − | + | </div> | |
| − | + | <div class="modal-body"> | |
| − | < | + | <ol> |
| − | + | <li class="licontent">Preculture the bacteria overnight by | |
| − | + | picking up a colony in 4ml LB with antibiotic.</li> | |
| − | + | <li class="licontent">Culture the bacteria in 30 ml LB by | |
| − | + | adding 1/100 of precultured broth, and selective antibiotic | |
| − | + | to log phase(about 3 hr).</li> | |
| − | + | <li class="licontent">Centrifuge them in 22℃ ,3200xg for 5 | |
| − | + | minutes , then refresh by the M9 medium with 4%xylose and | |
| − | + | 0.1%LB.</li> | |
| − | + | <li class="licontent">Culture the bacteria for 24 hr in both O<sub>2</sub> | |
| − | + | and 5%CO<sub>2</sub> incubator, and test the O.D. value at | |
| − | + | every 6 hours.</li> | |
| − | + | </ol> | |
| − | + | </div> | |
| − | + | <div class="modal-footer"> | |
| − | + | <button type="button" class="btn btn-secondary" data-dismiss="modal">Close</button> | |
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</div> | </div> | ||
</div> | </div> | ||
</div> | </div> | ||
| + | </div> | ||
| − | + | <div class="card col-md-4"> | |
| − | + | <div class="action-button" type="button" class="btn btn-primary" data-toggle="modal" | |
| − | + | data-target="#Carbonic_Anhydrase_Activity_Assay"> | |
| − | + | <div class="post"> | |
| − | + | <span class="folded-corner"></span> | |
| − | + | <img class="card-img-top" src="https://static.igem.org/mediawiki/2018/7/7b/T--NCKU_Tainan--protocol_Carbonic_anhydrase_activity_assay.jpg" | |
| − | + | alt="assay"> | |
| − | + | </div> | |
| − | + | <div class="card-body"> | |
| + | <h5 class="card-title">Carbonic Anhydrase Activity Assay</h5> | ||
</div> | </div> | ||
</div> | </div> | ||
| − | + | </div> | |
| − | + | <div class="modal" id="Carbonic_Anhydrase_Activity_Assay" tabindex="-1" role="dialog"> | |
| − | + | <div class="modal-dialog" role="document"> | |
| − | + | <div class="modal-content"> | |
| − | + | <div class="modal-header">Carbonic Anhydrase Activity Assay | |
| − | + | <button type="button" class="close" data-dismiss="modal" aria-label="Close"> | |
| − | + | <span aria-hidden="true">×</span> | |
| − | + | </button> | |
| − | + | </div> | |
| − | + | <div class="modal-body"> | |
| + | <h3>Materials:</h3> | ||
| + | <p class="blackp"> | ||
| + | pH meter, enzyme samples, magnetic stirrer, saturated CO2 | ||
| + | solution, 20 mM Tris-HCl | ||
| + | buffer (pH8.3), 20mL borosilicate glass vial | ||
| + | </p> | ||
| + | <h3>Method:</h3> | ||
| + | <ul> | ||
| + | <li class="licontent">Saturated CO2 solution preparation</li> | ||
| + | <p class="blackp">Dissolve gaseous CO2 into deionized water (on | ||
| + | ice) until it is saturated. (At least 30 min)</p> | ||
| + | <li class="licontent">20 mM Tris HCl buffer (pH8.3) preparation</li> | ||
<ol> | <ol> | ||
| − | <li class="licontent"> | + | <li class="licontent">Dissolve 121.14 g Tris in 800 ml |
| − | + | deionized water.</li> | |
| − | <li class="licontent"> | + | <li class="licontent">Add a pH meter into the solution to |
| − | + | observe the pH.</li> | |
| − | <li class="licontent"> | + | <li class="licontent">Slowly add concentrated hydrochloric |
| − | + | acid (HCl) solution to reduce the pH to | |
| − | + | 8.3. Be careful not to add too much at a time, since | |
| − | + | the pH will change rapidly.</li> | |
| − | + | <li class="licontent">Once the desired pH has been reached, | |
| − | <li class="licontent"> | + | top up the solution to 1 L using deionized water.</li> |
| − | + | ||
| − | + | ||
</ol> | </ol> | ||
| + | <li class="licontent">Performing Blank Reaction</li> | ||
| + | <ol start="5"> | ||
| + | <li class="licontent">Add 9 mL ice-cold Tris−HCl (20 mM, | ||
| + | pH8.3) buffer into a 20 mL borosilicate | ||
| + | glass vial with further incubation at 0 °C with | ||
| + | stirring.</li> | ||
| + | <li class="licontent">Add 6 mL of ice-cold CO2-saturated | ||
| + | solution immediately into the vial.</li> | ||
| + | <li class="licontent">Record the time course, T<sub>0</sub> | ||
| + | (in sec) of pH decrease from 8.3 to | ||
| + | 6.3. The pH meter must be preset to 0°C and calibrated.</li> | ||
| + | </ol> | ||
| + | <li class="licontent">Performing Test-Sample Reaction</li> | ||
| + | <ol start="8"> | ||
| + | <li class="licontent">Mix 9 mL ice-cold Tris−HCl (20 mM, | ||
| + | pH8.3) buffer and 0.2 mL enzyme, transfer | ||
| + | the mixture to a 20 mL borosilicate glass vial with | ||
| + | further incubation at 0 °C with stirring.</li> | ||
| + | <li class="licontent">Add 6 mL of ice-cold CO2-saturated | ||
| + | solution immediately into the vial.</li> | ||
| + | <li class="licontent">Record the time course, T (in sec) of | ||
| + | pH decrease from 8.3 to 6.3. The pH | ||
| + | meter must be preset to 0°C and calibrated.</li> | ||
| + | <li class="licontent">Calculate CA activity using a | ||
| + | Wilbur–Anderson unit (WAU) per milliliter of sample.</li> | ||
| + | </ol> | ||
| + | </ul> | ||
| + | <h3>Calculation:</h3> | ||
| + | <div> | ||
| + | <p class="blackp"> | ||
| + | Units/ml of enzyme = (T<sub>0 Average</sub> - T<sub>Average</sub> | ||
| + | ) (df) / (T<sub>Average</sub> )(E) | ||
| + | </p> | ||
</div> | </div> | ||
| − | <div class="modal-footer"> | + | <p class="blackp">T = Time (in seconds) required for pH to change |
| − | + | from 8.3 to 6.3 as per Unit Definition</p> | |
| − | + | <p class="blackp">df = dilution factor</p> | |
| + | <p class="blackp">E= volume (in milliliters) of enzyme used</p> | ||
| + | </div> | ||
| + | <div class="modal-footer"> | ||
| + | <button type="button" class="btn btn-secondary" data-dismiss="modal">Close</button> | ||
</div> | </div> | ||
</div> | </div> | ||
</div> | </div> | ||
</div> | </div> | ||
| − | |||
| − | + | <div class="card col-md-4"> | |
| − | + | <div class="action-button" type="button" class="btn btn-primary" data-toggle="modal" | |
| − | + | data-target="#Competent_Cell_Preparation"> | |
| − | + | <div class="post"> | |
| − | + | <span class="folded-corner"></span> | |
| − | + | <img class="card-img-top" src="https://static.igem.org/mediawiki/2018/3/38/T--NCKU_Tainan--protocol_competent.jpg" | |
| − | + | alt="Total solution"> | |
| + | </div> | ||
| + | <div class="card-body"> | ||
| + | <h5 class="card-title">Competent Cell Preparation</h5> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | <div class="modal" id="Competent_Cell_Preparation" tabindex="-1" role="dialog"> | ||
| + | <div class="modal-dialog" role="document"> | ||
| + | <div class="modal-content"> | ||
| + | <div class="modal-header">Competent Cell Preparation | ||
| + | <button type="button" class="close" data-dismiss="modal" aria-label="Close"> | ||
| + | <span aria-hidden="true">×</span> | ||
| + | </button> | ||
</div> | </div> | ||
| − | <div class=" | + | <div class="modal-body"> |
| − | < | + | <p class="blackp">For <i>E. coli</i> DH5α,BL21(DE3) and W3100(DE3) |
| + | competent cell</p> | ||
| + | <ol> | ||
| + | <li class="licontent">Streak out wild type <i>E. coli</i> on a | ||
| + | plate (LB plate without antibiotics) overnight | ||
| + | and pick one colony into 3 ml of media (LB) and grow | ||
| + | overnight.</li> | ||
| + | <li class="licontent">Transfer 0.4 ml of starter culture into | ||
| + | 40 ml of fresh LB and grow culture at 37 ℃.</li> | ||
| + | <li class="licontent">When the OD600 nm up to 0.35, put the | ||
| + | cells on ice immediately.</li> | ||
| + | <li class="licontent">Spin the cells at 4℃ for 10 minutes at | ||
| + | 4000 rpm.</li> | ||
| + | <li class="licontent">Suspend the pellet on ice carefully with | ||
| + | 16 ml chilly Transformation Buffer 1(TFB1).</li> | ||
| + | <li class="licontent">Leave nicely suspended bugs on ice for 10 | ||
| + | minutes.</li> | ||
| + | <li class="licontent">Spin the cells at 4℃ for 10 min. at 4000 | ||
| + | rpm.</li> | ||
| + | <li class="licontent">Suspend the pellet on ice with 1.6 ml of | ||
| + | Transformation Buffer 2 (TFB2).</li> | ||
| + | <li class="licontent">Leave on immediately on ice for 30 | ||
| + | minutes.</li> | ||
| + | <li class="licontent">Aliquot 100 μl into 1.5 ml centrifuge | ||
| + | tubes and snap freeze immediately with liquid nitrogen.</li> | ||
| + | <li class="licontent">Store the frozen cells in the -80℃ | ||
| + | freezer.</li> | ||
| + | </ol> | ||
| + | </div> | ||
| + | <div class="modal-footer"> | ||
| + | <button type="button" class="btn btn-secondary" data-dismiss="modal">Close</button> | ||
</div> | </div> | ||
</div> | </div> | ||
</div> | </div> | ||
| − | + | </div> | |
| − | + | </div> | |
| − | + | </div> | |
| − | + | ||
| − | + | <div class="row"> | |
| − | + | <div class="card-deck"> | |
| − | + | <div class="card col-md-4"> | |
| − | + | <div class="action-button" type="button" class="btn btn-primary" data-toggle="modal" | |
| − | + | data-target="#PCR"> | |
| − | + | <div class="post"> | |
| − | + | <span class="folded-corner"></span> | |
| − | + | <img class="card-img-top" src="https://static.igem.org/mediawiki/2018/0/01/T--NCKU_Tainan--protocol_PCR.jpg" | |
| − | + | alt="PCR"> | |
| − | + | </div> | |
| − | + | <div class="card-body"> | |
| − | + | <h5 class="card-title">PCR</h5> | |
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</div> | </div> | ||
</div> | </div> | ||
| − | + | </div> | |
| − | + | <div class="modal" id="PCR" tabindex="-1" role="dialog"> | |
| − | + | <div class="modal-dialog" role="document"> | |
| − | + | <div class="modal-content"> | |
| − | + | <div class="modal-header">PCR | |
| − | < | + | <button type="button" class="close" data-dismiss="modal" aria-label="Close"> |
| + | <span aria-hidden="true">×</span> | ||
| + | </button> | ||
</div> | </div> | ||
| − | <div class=" | + | <div class="modal-body"> |
| − | < | + | <ol> |
| + | <li class="licontent">Gently mix the following reaction by | ||
| + | pipetting and centrifuge briefly.</li> | ||
| + | <table class="centertable"> | ||
| + | <tr> | ||
| + | <th></th> | ||
| + | <th>20 μl system</th> | ||
| + | <th>50 μl system</th> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>Template</td> | ||
| + | <td>12～20 ng</td> | ||
| + | <td>30～50 ng</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>Forward primer</td> | ||
| + | <td>1.0 μl</td> | ||
| + | <td>2.5 μl</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>Reverse primer</td> | ||
| + | <td>1.0 μl</td> | ||
| + | <td>2.5 μl</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>dNTP</td> | ||
| + | <td>1.6 μl</td> | ||
| + | <td>4.0 μl</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>10x Buffer</td> | ||
| + | <td>2.0 μl</td> | ||
| + | <td>5.0 μl</td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | <p class="blackp">Program of KOD DNA polymerase</p> | ||
| + | <table class="centertable"> | ||
| + | <tr> | ||
| + | <th>Temperature</th> | ||
| + | <th>Time</th> | ||
| + | <th>Repeat</th> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>94 ℃</td> | ||
| + | <td>3 min.</td> | ||
| + | <td></td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>94 ℃</br>(Denaturation)</td> | ||
| + | <td>40 sec</td> | ||
| + | <td rowspan="3">25～30 cycles</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>57.5 ℃</br>(Annealing)</td> | ||
| + | <td>30 sec</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>72 ℃</br>(Extension)</td> | ||
| + | <td>Depend on sequence size</br>(2 kbp/min. for Taq)</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>72 ℃</td> | ||
| + | <td>5 min.</td> | ||
| + | <td></td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>4 ℃</td> | ||
| + | <td>∞</td> | ||
| + | <td></td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | <li class="licontent">Confirm the size of the digested product | ||
| + | by gel electrophoresis.</li> | ||
| + | <li class="licontent">Gel purification of the target size.</li> | ||
| + | </ol> | ||
</div> | </div> | ||
| − | + | <div class="modal-footer"> | |
| − | + | <button type="button" class="btn btn-secondary" data-dismiss="modal">Close</button> | |
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| − | + | ||
| − | + | ||
</div> | </div> | ||
</div> | </div> | ||
</div> | </div> | ||
| + | </div> | ||
| − | + | <div class="card col-md-4"> | |
| − | + | <div class="action-button" type="button" class="btn btn-primary" data-toggle="modal" | |
| − | + | data-target="#Plasmid_Construction"> | |
| − | + | <div class="post"> | |
| − | + | <span class="folded-corner"></span> | |
| − | + | <img class="card-img-top" src="https://static.igem.org/mediawiki/2018/4/41/T--NCKU_Tainan--protocol_plasmid.jpg" | |
| − | + | alt="Plasmid Construction"> | |
| − | + | </div> | |
| − | + | <div class="card-body"> | |
| + | <h5 class="card-title">Plasmid Construction</h5> | ||
</div> | </div> | ||
</div> | </div> | ||
| − | + | </div> | |
| − | + | <div class="modal" id="Plasmid_Construction" tabindex="-1" role="dialog"> | |
| − | + | <div class="modal-dialog" role="document"> | |
| − | + | <div class="modal-content"> | |
| − | + | <div class="modal-header">Plasmid Construction | |
| − | + | <button type="button" class="close" data-dismiss="modal" aria-label="Close"> | |
| − | + | <span aria-hidden="true">×</span> | |
| − | + | </button> | |
| − | + | </div> | |
| − | + | <div class="modal-body"> | |
| + | <div id="Plasmid_Construction"> | ||
<ol> | <ol> | ||
| − | <li class="licontent"> | + | <li class="licontent">Digestion (vector)</li> |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
</ol> | </ol> | ||
| − | < | + | <table class="centertable"> |
| + | <tr> | ||
| + | <th>Plasmid</th> | ||
| + | <th>200 ng</th> | ||
| + | <th>1000 ng</th> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>EcoRI-HF</td> | ||
| + | <td>0.2 μl</td> | ||
| + | <td>1 μl</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>SpeI-HF</td> | ||
| + | <td>0.2 μl</td> | ||
| + | <td>1 μl</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>CutSmart Buffer</td> | ||
| + | <td>2 μl</td> | ||
| + | <td>5 μl</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>ddH<sub>2</sub>O</td> | ||
| + | <td>17.6 μl</td> | ||
| + | <td>43 μl</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td><b>Total</b></td> | ||
| + | <td><b>20 μl</b></td> | ||
| + | <td><b>50 μl</b></td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>Digestion at 37℃ for 2.5hr</td> | ||
| + | <td></td> | ||
| + | <td></td> | ||
| + | </tr> | ||
| + | </table> | ||
<ol start="2"> | <ol start="2"> | ||
| − | <li class="licontent"> | + | <li class="licontent">Digestion (insert)</li> |
| − | + | ||
| − | + | ||
</ol> | </ol> | ||
| − | < | + | <table class="centertable"> |
| − | + | <tr> | |
| − | < | + | <th>Plasmid</th> |
| − | < | + | <th>200 ng</th> |
| − | < | + | <th>1000 ng</th> |
| − | < | + | </tr> |
| − | <li class="licontent"> | + | <tr> |
| − | <li class="licontent"> | + | <td>EcoRI-HF</td> |
| − | <li class="licontent"> | + | <td>0.2 μl</td> |
| + | <td>1 μl</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>XbaI</td> | ||
| + | <td>0.2 μl</td> | ||
| + | <td>1 μl</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>CutSmart Buffer</td> | ||
| + | <td>2 μl</td> | ||
| + | <td>5 μl</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>ddH<sub>2</sub>O</td> | ||
| + | <td>17.6 μl</td> | ||
| + | <td>43 μl</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td><b>Total</b></td> | ||
| + | <td><b>20 μl</b></td> | ||
| + | <td><b>50 μl</b></td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>Digestion at 37℃ for 2.5hr</td> | ||
| + | <td></td> | ||
| + | <td></td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | <ol start="3"> | ||
| + | <li class="licontent">Confirm the size of the digested | ||
| + | product by gel electrophoresis.</li> | ||
| + | <li class="licontent">Gel purification of the target size.</li> | ||
| + | <li class="licontent">Ligation</li> | ||
</ol> | </ol> | ||
| − | < | + | <table class="centertable"> |
| − | + | <tr> | |
| − | < | + | <th>Ingredient</th> |
| − | <li class="licontent"> | + | <th>Volume</th> |
| − | + | </tr> | |
| − | + | <tr> | |
| + | <td>Vector (2 kbp)</td> | ||
| + | <td rowspan="2">molar ratio = 1:3</br>(can be up to | ||
| + | 1:10, depends on the sizes of DNA)</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>Insert (1.5 kbp)</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>Quick Ligase Reaction Buffer (2X)*</td> | ||
| + | <td>10 μl</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>Quick Ligase</td> | ||
| + | <td>1 μl</td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>ddH<sub>2</sub>O</td> | ||
| + | <td>Up to 20 μl</td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | <ol start="6"> | ||
| + | <li class="licontent">Transform the product by heat shock.</li> | ||
</ol> | </ol> | ||
| − | |||
| − | |||
| − | |||
</div> | </div> | ||
| + | </div> | ||
| + | <div class="modal-footer"> | ||
| + | <button type="button" class="btn btn-secondary" data-dismiss="modal">Close</button> | ||
</div> | </div> | ||
</div> | </div> | ||
</div> | </div> | ||
| − | + | </div> | |
| − | + | ||
| − | + | <div class="card col-md-4"> | |
| − | + | <div class="action-button" type="button" class="btn btn-primary" data-toggle="modal" | |
| − | + | data-target="#PCR_Clean_Up"> | |
| − | + | <div class="post"> | |
| − | + | <span class="folded-corner"></span> | |
| − | + | <img class="card-img-top" src="https://static.igem.org/mediawiki/2018/3/34/T--NCKU_Tainan--protocol_PCR_cleanup.jpg" | |
| − | + | alt="Clean Up"> | |
| − | + | </div> | |
| − | + | <div class="card-body"> | |
| − | + | <h5 class="card-title">PCR Clean-Up & Gel Extraction</h5> | |
| − | + | ||
</div> | </div> | ||
</div> | </div> | ||
| − | + | </div> | |
| − | + | <div class="modal" id="PCR_Clean_Up" tabindex="-1" role="dialog"> | |
| − | + | <div class="modal-dialog" role="document"> | |
| − | + | <div class="modal-content"> | |
| − | + | <div class="modal-header">PCR Clean-Up & Gel Extraction | |
| − | + | <button type="button" class="close" data-dismiss="modal" aria-label="Close"> | |
| − | + | <span aria-hidden="true">×</span> | |
| − | + | </button> | |
| − | + | </div> | |
| + | <div class="modal-body"> | ||
| + | <h3>Gel Dissociation</h3> | ||
| + | <ol> | ||
| + | <li class="licontent">Gel Extraction</li> | ||
<ol> | <ol> | ||
| − | <li class="licontent"> | + | <li class="licontent">Excised the DNA fragment from the |
| − | + | agarose gel.</li> | |
| − | + | <li class="licontent">Transferred up to 300 mg of the gel | |
| − | <li class="licontent"> | + | slice to a 1.5 ml microcentrifuge tube.</li> |
| − | + | <li class="licontent">Added 500 μl of the Gel/PCR Bufffer | |
| − | + | to the sample and mixed by vortex.</li> | |
| − | + | <li class="licontent">Incubate at 55~60℃ for 10 minutes (or | |
| − | + | until the gel slice has completely dissolved).</li> | |
| − | + | <li class="licontent">During the incubation, mixed by | |
| − | + | vortexing the tube every 2~3 minutes.</li> | |
| − | + | <li class="licontent">Cooled the dissolved sample mixture | |
| − | + | to the room temperature.</li> | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | <li class="licontent"> | + | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | <li class="licontent"> | + | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
</ol> | </ol> | ||
| − | </ | + | </ol> |
| − | <div class="modal-footer"> | + | <h3>DNA Binding</h3> |
| − | + | <ol start="2"> | |
| − | + | <li class="licontent">Placed a PG Column in a Collection Tube. | |
| + | Apply the supernatant to the PG Column by decanting or | ||
| + | pipetting.</li> | ||
| + | <li class="licontent">Centrifuged at 16,000 xg for 30 seconds.</li> | ||
| + | <li class="licontent">Discarded the flow-through and place the | ||
| + | PG Column back into the same collection tube.</li> | ||
| + | </ol> | ||
| + | <h3>Wash</h3> | ||
| + | <ol start="5"> | ||
| + | <li class="licontent">Added 400 μl of the Buffer W1 into the PG | ||
| + | Column.</li> | ||
| + | <li class="licontent">Centrifuged at 16,000 xg for 30 seconds.</li> | ||
| + | <li class="licontent">Discarded the flow-through and place the | ||
| + | PG Column back into the same collection tube.</li> | ||
| + | <li class="licontent">Added 600 μl of the Buffer W2 (ethanol | ||
| + | added) into the PG Column.</li> | ||
| + | <li class="licontent">Centrifuged at 16,000 xg for 30 seconds.</li> | ||
| + | <li class="licontent">Discarded the flow-through and place the | ||
| + | PG Column back into the same collection tube.</li> | ||
| + | <li class="licontent">Centrifuged at 16,000 xg again for 2 | ||
| + | minutes to remove the residual Buffer W2.</li> | ||
| + | </ol> | ||
| + | <h3>Elution</h3> | ||
| + | <ol start="12"> | ||
| + | <li class="licontent">To elute the DNA, placed the PG Column in | ||
| + | a clean 1.5 ml microcentrifuge tube.</li> | ||
| + | <li class="licontent">Added 50 μl of the H<sub>2</sub>O (pH is | ||
| + | between 7.0 and 8.5) to the center of each PG | ||
| + | Column, let it stand for at least 2 minutes, and centrifuge | ||
| + | at 16,000 xg for 2 min. | ||
| + | </li> | ||
| + | </ol> | ||
| + | </div> | ||
| + | <div class="modal-footer"> | ||
| + | <button type="button" class="btn btn-secondary" data-dismiss="modal">Close</button> | ||
</div> | </div> | ||
</div> | </div> | ||
</div> | </div> | ||
| − | |||
| − | |||
</div> | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | |||
| + | <div class="row"> | ||
| + | <div class="card-deck"> | ||
| + | <div class="card col-md-4"> | ||
| + | <div class="action-button" type="button" class="btn btn-primary" data-toggle="modal" | ||
| + | data-target="#Plasmid_Extraction"> | ||
| + | <div class="post"> | ||
| + | <span class="folded-corner"></span> | ||
| + | <img class="card-img-top" src="https://static.igem.org/mediawiki/2018/1/1a/T--NCKU_Tainan--protocol_extraction.jpg" | ||
| + | alt="Extraction"> | ||
| + | </div> | ||
| + | <div class="card-body"> | ||
| + | <h5 class="card-title">Plasmid Extraction</h5> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | <div class="modal" id="Plasmid_Extraction" tabindex="-1" role="dialog"> | ||
| + | <div class="modal-dialog" role="document"> | ||
| + | <div class="modal-content"> | ||
| + | <div class="modal-header">Plasmid Extraction | ||
| + | <button type="button" class="close" data-dismiss="modal" aria-label="Close"> | ||
| + | <span aria-hidden="true">×</span> | ||
| + | </button> | ||
| + | </div> | ||
| + | <div class="modal-body"> | ||
| + | <ol> | ||
| + | <li class="licontent">Transfer 1.4 ml of well-grown bacterial | ||
| + | culture to a centrifuge tube.</li> | ||
| + | <li class="licontent">Centrifuge the tube at 16,000 xg for 1 | ||
| + | minute to pellet the cells and | ||
| + | discard the supernatant completely.</li> | ||
| + | <li class="licontent">Add 200 µl of FAPD1 Buffer (RNaseA added) | ||
| + | to the cell pellet and resuspend | ||
| + | the cells completely by pipetting.</li> | ||
| + | <ul> | ||
| + | <li class="licontent">Make sure that RNaseA has been added | ||
| + | into FAPD1 Buffer when first use.</li> | ||
| + | <li class="licontent">No cell pellet should be visible | ||
| + | after resuspension of the cells.</li> | ||
| + | </ul> | ||
| + | <li class="licontent">Add 200 µl of FAPD2 Buffer and gently | ||
| + | invert the tube 5 ~ 10 times. | ||
| + | Incubate the sample mixture at room temperature for 2 ~ 5 | ||
| + | minutes to lyse the cells. | ||
| + | </li> | ||
| + | <ul> | ||
| + | <li class="licontent">Do not vortex, vortex may shear | ||
| + | genomic DNA. If necessary, continue | ||
| + | inverting the tube until the lysate become clear.</li> | ||
| + | <li class="licontent">Make sure the tube transfer to | ||
| + | clarify from turbid.</li> | ||
| + | <li class="licontent">Do not proceed with the incubation | ||
| + | over 5 minutes.</li> | ||
| + | </ul> | ||
| + | <li class="licontent">Add 300 µl of FAPD3 Buffer and invert the | ||
| + | tube 5 ~ 10 times immediately to neutralize the lysate.</li> | ||
| + | <ul> | ||
| + | <li class="licontent">Invert immediately after adding FAPD3 | ||
| + | Buffer will avoid asymmetric precipitation.</li> | ||
| + | </ul> | ||
| + | <li class="licontent">Centrifuge at 16,000 xg for 3 minutess. | ||
| + | to clarify the lysate. During | ||
| + | centrifugation, place a FAPD Column in a Collection Tube.</li> | ||
| + | <li class="licontent">Transfer the supernatant carefully to the | ||
| + | FAPD Column and centrifuge at | ||
| + | 16,000 xg for 1 minute. Discard the flow-through and place | ||
| + | the column back to the Collection Tube.</li> | ||
| + | <ul> | ||
| + | <li class="licontent">Do not transfer any white pellet into | ||
| + | the column.</li> | ||
| + | </ul> | ||
| + | <li class="licontent">Add 400 µl of W1 Buffer to the FAPD | ||
| + | Column and centrifuge at 16,000 xg for 1 | ||
| + | minute. Discard the flow-through and place the column back | ||
| + | to the Collection Tube.</li> | ||
| + | <li class="licontent">Add 600 µl of Wash Buffer to the FAPD | ||
| + | Column and centrifuge at 16,000 xg for | ||
| + | 1 minute. Discard the flow-through and place the column | ||
| + | back to the Collection Tube.</li> | ||
| + | <ul> | ||
| + | <li class="licontent">Make sure that ethanol (96 ~ 100 %) | ||
| + | has been added into Wash Buffer when first use.</li> | ||
| + | </ul> | ||
| + | |||
| + | <li class="licontent">Centrifuge at 16,000 xg for an additional | ||
| + | 3 minutes to dry the FAPD Column.</li> | ||
| + | <ul> | ||
| + | <li class="licontent">Important step! The residual liquid | ||
| + | should be removed thoroughly on this step.</li> | ||
| + | </ul> | ||
| + | |||
| + | <li class="licontent">Place the FAPD Column to a new 1.5 ml | ||
| + | microcentrifuge tube.</li> | ||
| + | <li class="licontent">Add 30 µl of Elution Buffer or ddH<sub>2</sub>O | ||
| + | to the membrane center of the FAPD | ||
| + | Column. Stand the column for 3 minute. | ||
| + | </li> | ||
| + | <ul> | ||
| + | <li class="licontent">Important step! For effective | ||
| + | elution, make sure that the elution solution is | ||
| + | dispensed on the membrane center and is absorbed | ||
| + | completely.</li> | ||
| + | <li class="licontent">Do not elute the DNA using less than | ||
| + | suggested volume (50 µl). It will lower the final | ||
| + | yield.</li> | ||
| + | </ul> | ||
| + | <li class="licontent">Centrifuge at 16,000 xg for 3 minute to | ||
| + | elute plasmid DNA and store the DNA at -20 ℃. </li> | ||
| + | </ol> | ||
| + | </div> | ||
| + | <div class="modal-footer"> | ||
| + | <button type="button" class="btn btn-secondary" data-dismiss="modal">Close</button> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | <div class="card col-md-4 disappear" style="background-color: #272625; border-style: none;"></div> | ||
| + | <div class="card col-md-4 disappear" style="background-color: #272625; border-style: none;"></div> | ||
</div> | </div> | ||
</div> | </div> | ||
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{{NCKU_Tainan/footer}} | {{NCKU_Tainan/footer}} | ||
Latest revision as of 00:20, 3 November 2018
Protocol
Read
Dry Cell Weight
Minimal Salts (M9) Medium Xylose Preparation
PRK Toxicity Test
DNS Reductive Sugar Measurement
DNS Reductive Sugar Measurement
DNS solution preparation:
- Disolve 2.5g of 3,5-Dinitrosalicylic acid (DNS) to 150ml double distilled water.
- Heat to solution to 45 degree Celsius and add 4g of NaOH. Stir the solution until it is transparent.
- Add 75g of potassium sodium tartrate and add water to 250 ml.
- Keep the solution without light exposure. The solution can be used after 7 days.
Principle:
Under base solution, DNS will turn to brown color while reacting with reductive sugar in high temperature. In the specific temperature range, the color will have linear relationship with the reductive sugar concentration.
Calibration:
- Prepare glucose or xylose water solution to the following concentration: 0, 0.2, 0.4, 0.8, 1.0, 1.2, 1.6, 2 g/L.
- Take 200 ul of sample, add 200 ul of DNS solution.
- Heat the sample at 100 degree Celsius for 10mins.
- Cool down the sample with ice to room temperature.
- Measure the optical density of the sample with 540nm light wavelength.
- Draw the graph of sugar concentration with respect to optical density.
Calibration results:
Measurement:
- Take 200 ul of sample, add 200 ul of DNS solution.
- Heat the sample at 100 degree Celsius for 10mins.
- Cool down the sample with ice to room temperature.
- Measure the optical density of the sample with 540nm light wavelength.
- Get the concentration of reductive sugar from the Calibration graph.
Short Term pH Sensing Systen Measurement
Long Term pH Sensing System Measurement
Total Solution
Carbonic Anhydrase Activity Assay
Competent Cell Preparation
PCR
Plasmid Construction
PCR Clean-Up & Gel Extraction
