|
|
| Line 24: |
Line 24: |
| | </div> | | </div> |
| | <div class="card-body"> | | <div class="card-body"> |
| − | <h5 class="card-title">Dry cell weight</h5> | + | <h5 class="card-title">Dry Cell Weight</h5> |
| | </div> | | </div> |
| | </div> | | </div> |
| Line 31: |
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| | <div class="modal-dialog" role="document"> | | <div class="modal-dialog" role="document"> |
| | <div class="modal-content"> | | <div class="modal-content"> |
| − | <div class="modal-header">Dry cell weight | + | <div class="modal-header">Dry Cell Weight |
| | <button type="button" class="close" data-dismiss="modal" aria-label="Close"> | | <button type="button" class="close" data-dismiss="modal" aria-label="Close"> |
| | <span aria-hidden="true">×</span> | | <span aria-hidden="true">×</span> |
| Line 63: |
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| | </div> | | </div> |
| | <div class="card-body"> | | <div class="card-body"> |
| − | <h5 class="card-title">Minimal salts (M9) Medium Xylose Preparation</h5> | + | <h5 class="card-title">Minimal Salts (M9) Medium Xylose Preparation</h5> |
| | </div> | | </div> |
| | </div> | | </div> |
| Line 70: |
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| | <div class="modal-dialog" role="document"> | | <div class="modal-dialog" role="document"> |
| | <div class="modal-content"> | | <div class="modal-content"> |
| − | <div class="modal-header">Minimal salts (M9) Medium Xylose Preparation | + | <div class="modal-header">Minimal Salts (M9) Medium Xylose Preparation |
| | <button type="button" class="close" data-dismiss="modal" aria-label="Close"> | | <button type="button" class="close" data-dismiss="modal" aria-label="Close"> |
| | <span aria-hidden="true">×</span> | | <span aria-hidden="true">×</span> |
| Line 170: |
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| | <div class="post"> | | <div class="post"> |
| | <span class="folded-corner"></span> | | <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"> | + | <img class="card-img-top" src="https://static.igem.org/mediawiki/2018/d/d9/T--NCKU_Tainan--protocol_PRK_Toxicity_Test.jpg" alt="PRK Toxicity Test"> |
| | </div> | | </div> |
| | <div class="card-body"> | | <div class="card-body"> |
| Line 207: |
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| | <div class="action-button" type="button" class="btn btn-primary" data-toggle="modal" data-target="#DNS_Reductive_Sugar_Measurement"> | | <div class="action-button" type="button" class="btn btn-primary" data-toggle="modal" data-target="#DNS_Reductive_Sugar_Measurement"> |
| | <div class="post"> | | <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="DNS">
| |
| − | </div>
| |
| − | <div class="card-body">
| |
| − | <h5 class="card-title">DNS Reductive Sugar Measurement</h5>
| |
| − | </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>
| |
| − | </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 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 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">
| |
| − | <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 alert systen measurement</h5>
| |
| − | </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 alert systen 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
| |
| − | (pH4,4.25,4.5,4.75,5,5.5,6,7) pH value of M9 buffer.</li>
| |
| − | <li class="licontent">Culture the bacteria in 6 ml LB with 1/1000 chloramphenicol to log phase (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 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">
| |
| − | <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 alert system measurement</h5>
| |
| − | </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 alert 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
| |
| − | (pH4,4.25,4.5,4.75,5,5.5,6,7) 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
| |
| − | 1/1000 chloramphenicol.</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 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 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, 1/2000
| |
| − | kanamycin, 1/4000 chloramphenicol 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 (130 rpm), 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>
| |
| − | </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 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>
| |
| − | <li class="licontent">Dissolve 121.14 g Tris in 800 ml deionized water.</li>
| |
| − | <li class="licontent">Add a pH meter into the solution to observe the pH.</li>
| |
| − | <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, top up the solution to 1 L using deionized water.</li>
| |
| − | </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>
| |
| − | <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 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 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 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>
| |
| − | </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 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 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">
| |
| − | <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 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>
| |
| − | <li class="licontent">Digestion (vector)</li>
| |
| − | </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>Up to 20 μl</td>
| |
| − | <td>Up to 50 μl</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Digestion at 37℃ for 2.5hr.</td>
| |
| − | <td></td>
| |
| − | <td></td>
| |
| − | </tr>
| |
| − | </table>
| |
| − | <ol start="2">
| |
| − | <li class="licontent">Digestion (insert)</li>
| |
| − | </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>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>Up to 20 μl</td>
| |
| − | <td>Up to 50 μl</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>
| |
| − | <table class="centertable">
| |
| − | <tr>
| |
| − | <th>Ingredient</th>
| |
| − | <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>
| |
| − | </div>
| |
| − | </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">
| |
| − | <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 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>
| |
| − | <li class="licontent">Excised the DNA fragment from the agarose gel.</li>
| |
| − | <li class="licontent">Transferred up to 300 mg of the gel 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>
| |
| − | </ol>
| |
| − | </ol>
| |
| − | <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 H2O (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 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 ddH2O 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>
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