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<div class="menu"> | <div class="menu"> | ||
<a class="item" href="https://2016.igem.org/Team:USTC/Team"> Members </a> | <a class="item" href="https://2016.igem.org/Team:USTC/Team"> Members </a> | ||
− | <a class="item" href="https://2016.igem.org/Team:USTC/Team/Sponsor"> Sponsor </a> | + | <a class="item" href="https://2016.igem.org/Team:USTC/Team/Sponsor"> Sponsor </a> |
− | <a class="item" href="https://2016.igem.org/Team:USTC/Team/AboutUSTC"> About USTC </a> | + | <a class="item" href="https://2016.igem.org/Team:USTC/Team/AboutUSTC"> About USTC </a> |
− | <a class="item" href="https://2016.igem.org/Team:USTC/Team/Acknowledgements"> Acknowledgements </a> | + | <a class="item" href="https://2016.igem.org/Team:USTC/Team/Acknowledgements"> Acknowledgements </a> |
− | <a class="item" href="https://2016.igem.org/Team:USTC/Team/Gallery"> Gallery </a> | + | <a class="item" href="https://2016.igem.org/Team:USTC/Team/Gallery"> Gallery </a> |
− | <a class="item" href="https://2016.igem.org/Team:USTC/Collaborations"> Collaborations </a> | + | <a class="item" href="https://2016.igem.org/Team:USTC/Collaborations"> Collaborations </a> |
</div> | </div> | ||
</div> | </div> | ||
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<div class="menu"> | <div class="menu"> | ||
<a class="item" href="https://2016.igem.org/Team:USTC/Team"> Members </a> | <a class="item" href="https://2016.igem.org/Team:USTC/Team"> Members </a> | ||
− | <a class="item" href="https://2016.igem.org/Team:USTC/Team/Sponsor"> Sponsor </a> | + | <a class="item" href="https://2016.igem.org/Team:USTC/Team/Sponsor"> Sponsor </a> |
− | <a class="item" href="https://2016.igem.org/Team:USTC/Team/AboutUSTC"> About USTC </a> | + | <a class="item" href="https://2016.igem.org/Team:USTC/Team/AboutUSTC"> About USTC </a> |
− | <a class="item" href="https://2016.igem.org/Team:USTC/Team/Acknowledgements"> Acknowledgements </a> | + | <a class="item" href="https://2016.igem.org/Team:USTC/Team/Acknowledgements"> Acknowledgements </a> |
− | <a class="item" href="https://2016.igem.org/Team:USTC/Team/Gallery"> Gallery </a> | + | <a class="item" href="https://2016.igem.org/Team:USTC/Team/Gallery"> Gallery </a> |
− | <a class="item" href="https://2016.igem.org/Team:USTC/Collaborations"> Collaborations </a> | + | <a class="item" href="https://2016.igem.org/Team:USTC/Collaborations"> Collaborations </a> |
</div> | </div> | ||
</div> | </div> | ||
Line 260: | Line 260: | ||
− | <p><strong>DATE:8.24</strong></p> | + | <p><strong>DATE:8.24</strong></p> |
− | <p><strong>Recorder: Yu Xie</strong> | + | <p><strong>Recorder: Yu Xie</strong> |
− | <strong>Plasmid Extraction for sfGFP11</strong></p> | + | <strong>Plasmid Extraction for sfGFP11</strong></p> |
− | <p><strong>Recorder: Yu Xie</strong> | + | <p><strong>Recorder: Yu Xie</strong> |
− | <strong>PCR of sup35</strong></p> | + | <strong>PCR of sup35</strong></p> |
− | <p>Procedure:</p> | + | <p>Procedure:</p> |
− | <p>1.Prepare 8 PCR tubes and sequentially add:</p> | + | <p>1.Prepare 8 PCR tubes and sequentially add:</p> |
− | <table class="ui celled small table"> | + | <table class="ui celled small table"> |
− | <thead> | + | <thead> |
− | <tr> | + | <tr> |
− | + | <th>sample</th> | |
− | + | <th>1,2,3,4,5,6,7,8</th> | |
− | </tr> | + | </tr> |
− | </thead> | + | </thead> |
− | <tbody> | + | <tbody> |
− | <tr> | + | <tr> |
− | + | <td>Sterilized ddH<sub>2</sub>O</td> | |
− | + | <td>9 μL</td> | |
− | </tr> | + | </tr> |
− | <tr> | + | <tr> |
− | + | <td>2×HiFi-PCR Master</td> | |
− | + | <td>12.5 μL</td> | |
− | </tr> | + | </tr> |
− | <tr> | + | <tr> |
− | + | <td>Template</td> | |
− | + | <td>1.5 μL</td> | |
− | </tr> | + | </tr> |
− | <tr> | + | <tr> |
− | + | <td>Primer 1</td> | |
− | + | <td>1 μL</td> | |
− | </tr> | + | </tr> |
− | <tr> | + | <tr> |
− | + | <td>Primer 2</td> | |
− | + | <td>1 μL</td> | |
− | </tr> | + | </tr> |
− | <tr> | + | <tr> |
− | + | <td>total</td> | |
− | + | <td>25 μL</td> | |
− | </tr> | + | </tr> |
− | </tbody> | + | </tbody> |
− | <table class="ui celled small table"> | + | <table class="ui celled small table"> |
− | <p>2.PCR reaction | + | <p>2.PCR reaction |
− | Parameters setting:</p> | + | Parameters setting:</p> |
− | <table class="ui celled small table"> | + | <table class="ui celled small table"> |
− | <thead> | + | <thead> |
− | <tr> | + | <tr> |
− | + | <th>stage</th> | |
− | + | <th>temperature</th> | |
− | + | <th>time</th> | |
− | </tr> | + | </tr> |
− | </thead> | + | </thead> |
− | <tbody> | + | <tbody> |
− | <tr> | + | <tr> |
− | + | <td>Pre-Duration</td> | |
− | + | <td>95℃</td> | |
− | + | <td>10 min</td> | |
− | </tr> | + | </tr> |
− | <tr> | + | <tr> |
− | + | <td>Duration</td> | |
− | + | <td>95℃</td> | |
− | + | <td>1 min</td> | |
− | </tr> | + | </tr> |
− | <tr> | + | <tr> |
− | + | <td>Anneal</td> | |
− | + | <td>58℃</td> | |
− | + | <td>1 min</td> | |
− | </tr> | + | </tr> |
− | <tr> | + | <tr> |
− | + | <td>Extend</td> | |
− | + | <td>72℃</td> | |
− | + | <td>1 min 50 s</td> | |
− | </tr> | + | </tr> |
− | <tr> | + | <tr> |
− | + | <td>Post-Extend</td> | |
− | + | <td>72℃</td> | |
− | + | <td>10 min</td> | |
− | </tr> | + | </tr> |
− | <tr> | + | <tr> |
− | + | <td>Final</td> | |
− | + | <td>4℃</td> | |
− | + | <td>--</td> | |
− | </tr> | + | </tr> |
− | </tbody> | + | </tbody> |
− | <table class="ui celled small table"> | + | <table class="ui celled small table"> |
− | <p>30 cycles(Duration ~ Extend)</p> | + | <p>30 cycles(Duration ~ Extend)</p> |
− | <p><strong>Recorder: Yu Xie</strong> | + | <p><strong>Recorder: Yu Xie</strong> |
− | <strong>Double digestion of sup35</strong></p> | + | <strong>Double digestion of sup35</strong></p> |
− | <p><strong>Recorder:Yu Xie</strong> | + | <p><strong>Recorder:Yu Xie</strong> |
− | <strong>Ligation of sup35 and pUC57-sfGFP11</strong></p> | + | <strong>Ligation of sup35 and pUC57-sfGFP11</strong></p> |
− | <p><strong>Recorder: Yu Xie</strong> | + | <p><strong>Recorder: Yu Xie</strong> |
− | <strong>Transformation of combinant plasmid pUC57 containing sup35-sfGFP11</strong></p> | + | <strong>Transformation of combinant plasmid pUC57 containing sup35-sfGFP11</strong></p> |
− | <p><strong>Recorder: Yu Xie</strong> | + | <p><strong>Recorder: Yu Xie</strong> |
− | <strong>PCR of sup35</strong></p> | + | <strong>PCR of sup35</strong></p> |
− | <p>Procedure:</p> | + | <p>Procedure:</p> |
− | <p>1.Prepare 8 PCR tubes and sequentially add:</p> | + | <p>1.Prepare 8 PCR tubes and sequentially add:</p> |
− | <table class="ui celled small table"> | + | <table class="ui celled small table"> |
− | <thead> | + | <thead> |
− | <tr> | + | <tr> |
− | + | <th>sample</th> | |
− | + | <th>1,2,3,4,5,6,7,8</th> | |
− | </tr> | + | </tr> |
− | </thead> | + | </thead> |
− | <tbody> | + | <tbody> |
− | <tr> | + | <tr> |
− | + | <td>Sterilized ddH<sub>2</sub>O</td> | |
− | + | <td>9 μL</td> | |
− | </tr> | + | </tr> |
− | <tr> | + | <tr> |
− | + | <td>2×HiFi-PCR Master</td> | |
− | + | <td>12.5 μL</td> | |
− | </tr> | + | </tr> |
− | <tr> | + | <tr> |
− | + | <td>Template</td> | |
− | + | <td>1.5 μL</td> | |
− | </tr> | + | </tr> |
− | <tr> | + | <tr> |
− | + | <td>Primer 1</td> | |
− | + | <td>1 μL</td> | |
− | </tr> | + | </tr> |
− | <tr> | + | <tr> |
− | + | <td>Primer 2</td> | |
− | + | <td>1 μL</td> | |
− | </tr> | + | </tr> |
− | <tr> | + | <tr> |
− | + | <td>total</td> | |
− | + | <td>25 μL</td> | |
− | </tr> | + | </tr> |
− | </tbody> | + | </tbody> |
− | <table class="ui celled small table"> | + | <table class="ui celled small table"> |
− | <p>2.PCR reaction | + | <p>2.PCR reaction |
− | Parameters setting:</p> | + | Parameters setting:</p> |
− | <table class="ui celled small table"> | + | <table class="ui celled small table"> |
− | <thead> | + | <thead> |
− | <tr> | + | <tr> |
− | + | <th>stage</th> | |
− | + | <th>temperature</th> | |
− | + | <th>time</th> | |
− | </tr> | + | </tr> |
− | </thead> | + | </thead> |
− | <tbody> | + | <tbody> |
− | <tr> | + | <tr> |
− | + | <td>Pre-Duration</td> | |
− | + | <td>95℃</td> | |
− | + | <td>10 min</td> | |
− | </tr> | + | </tr> |
− | <tr> | + | <tr> |
− | + | <td>Duration</td> | |
− | + | <td>95℃</td> | |
− | + | <td>1 min</td> | |
− | </tr> | + | </tr> |
− | <tr> | + | <tr> |
− | + | <td>Anneal</td> | |
− | + | <td>58℃</td> | |
− | + | <td>1 min</td> | |
− | </tr> | + | </tr> |
− | <tr> | + | <tr> |
− | + | <td>Extend</td> | |
− | + | <td>72℃</td> | |
− | + | <td>1 min 50s</td> | |
− | </tr> | + | </tr> |
− | <tr> | + | <tr> |
− | + | <td>Post-Extend</td> | |
− | + | <td>72℃</td> | |
− | + | <td>10 min</td> | |
− | </tr> | + | </tr> |
− | <tr> | + | <tr> |
− | + | <td>Final</td> | |
− | + | <td>4℃</td> | |
− | + | <td>--</td> | |
− | </tr> | + | </tr> |
− | </tbody> | + | </tbody> |
− | <table class="ui celled small table"> | + | <table class="ui celled small table"> |
− | <p>30 cycles(Duration ~ Extend)</p> | + | <p>30 cycles(Duration ~ Extend)</p> |
− | <p><strong>Recorder:Yu Xie</strong> | + | <p><strong>Recorder:Yu Xie</strong> |
− | <strong>Colony picking of sfGFP11</strong></p> | + | <strong>Colony picking of sfGFP11</strong></p> |
− | <p><strong>Recorder: Chenyang Li</strong> | + | <p><strong>Recorder: Chenyang Li</strong> |
− | <strong>PCR of pYeWGAP-BD</strong></p> | + | <strong>PCR of pYeWGAP-BD</strong></p> |
− | <p><strong>Procedure:</strong></p> | + | <p><strong>Procedure:</strong></p> |
− | <p>3.Prepare 4 PCR of pYeWGAP-BD tubes and sequentially add: | + | <p>3.Prepare 4 PCR of pYeWGAP-BD tubes and sequentially add: |
− | + | 4 μL Sterilized ddH<sub>2</sub>O, | |
− | + | 6.5 μL 2XHiFi Pfu, | |
− | + | 0.5 μL primer F, | |
− | + | 0.5 μL primer R, | |
− | + | 1 μL DNA template.</p> | |
− | <p>4.PCR reaction | + | <p>4.PCR reaction |
− | Parameters setting:</p> | + | Parameters setting:</p> |
− | <table class="ui celled small table"> | + | <table class="ui celled small table"> |
− | <thead> | + | <thead> |
− | <tr> | + | <tr> |
− | + | <th>stage</th> | |
− | + | <th>temperature</th> | |
− | + | <th>time</th> | |
− | </tr> | + | </tr> |
− | </thead> | + | </thead> |
− | <tbody> | + | <tbody> |
− | <tr> | + | <tr> |
− | + | <td>step 1</td> | |
− | + | <td>95℃</td> | |
− | + | <td>10 min</td> | |
− | </tr> | + | </tr> |
− | <tr> | + | <tr> |
− | + | <td>step 2</td> | |
− | + | <td>95℃</td> | |
− | + | <td>30 s</td> | |
− | </tr> | + | </tr> |
− | <tr> | + | <tr> |
− | + | <td>step 3</td> | |
− | + | <td>58℃</td> | |
− | + | <td>45 s</td> | |
− | </tr> | + | </tr> |
− | <tr> | + | <tr> |
− | + | <td>step 4</td> | |
− | + | <td>72℃</td> | |
− | + | <td>45 s</td> | |
− | </tr> | + | </tr> |
− | <tr> | + | <tr> |
− | + | <td>step 5</td> | |
− | + | <td>72℃</td> | |
− | + | <td>10 min</td> | |
− | </tr> | + | </tr> |
− | <tr> | + | <tr> |
− | + | <td>step 6</td> | |
− | + | <td>4℃</td> | |
− | + | <td>--</td> | |
− | </tr> | + | </tr> |
− | </tbody> | + | </tbody> |
− | <table class="ui celled small table"> | + | <table class="ui celled small table"> |
− | <p>30 cycles(step 2 ~ step 4)</p> | + | <p>30 cycles(step 2 ~ step 4)</p> |
− | <p>results:</p> | + | <p>results:</p> |
− | <p><img src="https://static.igem.org/mediawiki/2016/thumb/4/45/T--USTC--NB_0823_1.jpeg/750px-T--USTC--NB_0823_1.jpeg" alt="a" /> | + | <p><img src="https://static.igem.org/mediawiki/2016/thumb/4/45/T--USTC--NB_0823_1.jpeg/750px-T--USTC--NB_0823_1.jpeg" alt="a" /> |
− | (from left to right: Trans 2K plus 2(contain Gelred), 1, 2, 3, 4) | + | (from left to right: Trans 2K plus 2(contain Gelred), 1, 2, 3, 4) |
− | </p> | + | </p> |
− | <p><strong>Plasmid Extraction for ligased YeWGAP and BD</strong> | + | <p><strong>Plasmid Extraction for ligased YeWGAP and BD</strong> |
− | <strong>Recorder: Chenyang Li</strong></p> | + | <strong>Recorder: Chenyang Li</strong></p> |
− | <p><strong>results</strong></p> | + | <p><strong>results</strong></p> |
− | <table class="ui celled small table"> | + | <table class="ui celled small table"> |
− | <thead> | + | <thead> |
− | <tr> | + | <tr> |
− | + | <th>sample</th> | |
− | + | <th>YBE8241001</th> | |
− | + | <th>YBE8241002</th> | |
− | + | <th>YBE8241003</th> | |
− | + | <th>YBE8241004</th> | |
− | </tr> | + | </tr> |
− | </thead> | + | </thead> |
− | <tbody> | + | <tbody> |
− | <tr> | + | <tr> |
− | + | <td>A260/A280</td> | |
− | + | <td>1.88</td> | |
− | + | <td>1.86</td> | |
− | + | <td>1.89</td> | |
− | + | <td>1.88</td> | |
− | </tr> | + | </tr> |
− | <tr> | + | <tr> |
− | + | <td>A260/A230</td> | |
− | + | <td>2.27</td> | |
− | + | <td>1.92</td> | |
− | + | <td>2.22</td> | |
− | + | <td>2.22</td> | |
− | </tr> | + | </tr> |
− | <tr> | + | <tr> |
− | + | <td>concentration(ng/μL)</td> | |
− | + | <td>203.1</td> | |
− | + | <td>174.7</td> | |
− | + | <td>181.3</td> | |
− | + | <td>177.8</td> | |
− | </tr> | + | </tr> |
− | </tbody> | + | </tbody> |
− | <table class="ui celled small table"> | + | <table class="ui celled small table"> |
− | <p><strong>DATE:8.24</strong> | + | <p><strong>DATE:8.24</strong> |
− | <strong>Recorder: Chenyang Li</strong> | + | <strong>Recorder: Chenyang Li</strong> |
− | <strong>PCR of pYeWGAP-BD</strong></p> | + | <strong>PCR of pYeWGAP-BD</strong></p> |
− | <p><strong>Procedure:</strong></p> | + | <p><strong>Procedure:</strong></p> |
− | <p>3.Prepare 4 PCR of pYeWGAP-BD tubes and sequentially add: | + | <p>3.Prepare 4 PCR of pYeWGAP-BD tubes and sequentially add: |
− | 3 μL Sterilized ddH<sub>2</sub>O, | + | 3 μL Sterilized ddH<sub>2</sub>O, |
− | + | 5 μL 2XHiFi Pfu, | |
− | + | 0.5 μL primer F, | |
− | + | 0.5 μL primer R, | |
− | + | 1 μL DNA template.</p> | |
− | <p>4.PCR reaction | + | <p>4.PCR reaction |
− | Parameters setting:</p> | + | Parameters setting:</p> |
− | <table class="ui celled small table"> | + | <table class="ui celled small table"> |
− | <thead> | + | <thead> |
− | <tr> | + | <tr> |
− | + | <th>stage</th> | |
− | + | <th>temperature</th> | |
− | + | <th>time</th> | |
− | </tr> | + | </tr> |
− | </thead> | + | </thead> |
− | <tbody> | + | <tbody> |
− | <tr> | + | <tr> |
− | + | <td>step 1</td> | |
− | + | <td>95℃</td> | |
− | + | <td>10 min</td> | |
− | </tr> | + | </tr> |
− | <tr> | + | <tr> |
− | + | <td>step 2</td> | |
− | + | <td>95℃</td> | |
− | + | <td>30 s</td> | |
− | </tr> | + | </tr> |
− | <tr> | + | <tr> |
− | + | <td>step 3</td> | |
− | + | <td>58℃</td> | |
− | + | <td>45 s</td> | |
− | </tr> | + | </tr> |
− | <tr> | + | <tr> |
− | + | <td>step 4</td> | |
− | + | <td>72℃</td> | |
− | + | <td>45 s</td> | |
− | </tr> | + | </tr> |
− | <tr> | + | <tr> |
− | + | <td>step 5</td> | |
− | + | <td>72℃</td> | |
− | + | <td>10 min</td> | |
− | </tr> | + | </tr> |
− | <tr> | + | <tr> |
− | + | <td>step 6</td> | |
− | + | <td>4℃</td> | |
− | + | <td>--</td> | |
− | </tr> | + | </tr> |
− | </tbody> | + | </tbody> |
− | <table class="ui celled small table"> | + | <table class="ui celled small table"> |
− | <p>30 cycles(step 2 ~ step 4)</p> | + | <p>30 cycles(step 2 ~ step 4)</p> |
− | <p>results:</p> | + | <p>results:</p> |
− | <p><img src="https://static.igem.org/mediawiki/2016/thumb/5/54/T--USTC--NB_0820_2.jpeg/750px-T--USTC--NB_0820_2.jpeg" alt="a" /> | + | <p><img src="https://static.igem.org/mediawiki/2016/thumb/5/54/T--USTC--NB_0820_2.jpeg/750px-T--USTC--NB_0820_2.jpeg" alt="a" /> |
− | (from left to right: Trans 2K plus 2(contain Gelred),YBE8241001,1,YBE8241002,2,YBE8241003,3,YBE8241004,4,) | + | (from left to right: Trans 2K plus 2(contain Gelred),YBE8241001,1,YBE8241002,2,YBE8241003,3,YBE8241004,4,) |
− | </p> | + | </p> |
− | <p><strong>DATE:8.25</strong></p> | + | <p><strong>DATE:8.25</strong></p> |
− | <p><strong>Recorder: Yu Xie</strong> | + | <p><strong>Recorder: Yu Xie</strong> |
− | <strong>Plasmid Extraction for sfGFP11</strong></p> | + | <strong>Plasmid Extraction for sfGFP11</strong></p> |
− | <p><strong>Recorder: Yu Xie</strong> | + | <p><strong>Recorder: Yu Xie</strong> |
− | <strong>Transformation of combinant plasmid pUC57 containing sup35-sfGFP11</strong></p> | + | <strong>Transformation of combinant plasmid pUC57 containing sup35-sfGFP11</strong></p> |
− | <p><strong>Recorder: Yu Xie</strong> | + | <p><strong>Recorder: Yu Xie</strong> |
− | <strong>PCR of sup35</strong></p> | + | <strong>PCR of sup35</strong></p> |
− | <p>Procedure:</p> | + | <p>Procedure:</p> |
− | <p>1.Prepare 8 PCR tubes and sequentially add:</p> | + | <p>1.Prepare 8 PCR tubes and sequentially add:</p> |
− | <table class="ui celled small table"> | + | <table class="ui celled small table"> |
− | <thead> | + | <thead> |
− | <tr> | + | <tr> |
− | + | <th>sample</th> | |
− | + | <th>1,2,3,4,5,6,7,8</th> | |
− | </tr> | + | </tr> |
− | </thead> | + | </thead> |
− | <tbody> | + | <tbody> |
− | <tr> | + | <tr> |
− | + | <td>Sterilized ddH<sub>2</sub>O</td> | |
− | + | <td>9 μL</td> | |
− | </tr> | + | </tr> |
− | <tr> | + | <tr> |
− | + | <td>2×HiFi-PCR Master</td> | |
− | + | <td>12.5 μL</td> | |
− | </tr> | + | </tr> |
− | <tr> | + | <tr> |
− | + | <td>Template</td> | |
− | + | <td>1.5 μL</td> | |
− | </tr> | + | </tr> |
− | <tr> | + | <tr> |
− | + | <td>Primer 1</td> | |
− | + | <td>1 μL</td> | |
− | </tr> | + | </tr> |
− | <tr> | + | <tr> |
− | + | <td>Primer 2</td> | |
− | + | <td>1 μL</td> | |
− | </tr> | + | </tr> |
− | <tr> | + | <tr> |
− | + | <td>total</td> | |
− | + | <td>25 μL</td> | |
− | </tr> | + | </tr> |
− | </tbody> | + | </tbody> |
− | <table class="ui celled small table"> | + | <table class="ui celled small table"> |
− | <p>2.PCR reaction | + | <p>2.PCR reaction |
− | Parameters setting:</p> | + | Parameters setting:</p> |
− | <table class="ui celled small table"> | + | <table class="ui celled small table"> |
− | <thead> | + | <thead> |
− | <tr> | + | <tr> |
− | + | <th>stage</th> | |
− | + | <th>temperature</th> | |
− | + | <th>time</th> | |
− | </tr> | + | </tr> |
− | </thead> | + | </thead> |
− | <tbody> | + | <tbody> |
− | <tr> | + | <tr> |
− | + | <td>Pre-Duration</td> | |
− | + | <td>95℃</td> | |
− | + | <td>10 min</td> | |
− | </tr> | + | </tr> |
− | <tr> | + | <tr> |
− | + | <td>Duration</td> | |
− | + | <td>95℃</td> | |
− | + | <td>1 min</td> | |
− | </tr> | + | </tr> |
− | <tr> | + | <tr> |
− | + | <td>Anneal</td> | |
− | + | <td>58℃</td> | |
− | + | <td>1 min</td> | |
− | </tr> | + | </tr> |
− | <tr> | + | <tr> |
− | + | <td>Extend</td> | |
− | + | <td>72℃</td> | |
− | + | <td>1 min 50 s</td> | |
− | </tr> | + | </tr> |
− | <tr> | + | <tr> |
− | + | <td>Post-Extend</td> | |
− | + | <td>72℃</td> | |
− | + | <td>10 min</td> | |
− | </tr> | + | </tr> |
− | <tr> | + | <tr> |
− | + | <td>Final</td> | |
− | + | <td>4℃</td> | |
− | + | <td>--</td> | |
− | </tr> | + | </tr> |
− | </tbody> | + | </tbody> |
− | <table class="ui celled small table"> | + | <table class="ui celled small table"> |
− | <p>30 cycles(Duration ~ Extend)</p> | + | <p>30 cycles(Duration ~ Extend)</p> |
− | <p><strong>Recorder:Yu Xie</strong> | + | <p><strong>Recorder:Yu Xie</strong> |
− | <strong>Colony picking of sup35-sfGFP11</strong></p> | + | <strong>Colony picking of sup35-sfGFP11</strong></p> |
− | <p><strong>Ligation of YeWGAP and BD</strong> | + | <p><strong>Ligation of YeWGAP and BD</strong> |
− | **Recorder: Chenyang Li **</p> | + | **Recorder: Chenyang Li **</p> |
− | <p>Procedure: | + | <p>Procedure: |
− | 9 μL YeWGAPD8201002 | + | 9 μL YeWGAPD8201002 |
− | 3.3 μL BDD8201002 | + | 3.3 μL BDD8201002 |
− | 0.2 μL T4 DNA ligase | + | 0.2 μL T4 DNA ligase |
− | 2 μL 10×T4 DNA Ligase Buffer | + | 2 μL 10×T4 DNA Ligase Buffer |
− | 5.5 μL ddH<sub>2</sub>O | + | 5.5 μL ddH<sub>2</sub>O |
− | 20 μL in total.</p> | + | 20 μL in total.</p> |
− | <p>Mix gently and incubate at 22 degree Celsius for an hour.</p> | + | <p>Mix gently and incubate at 22 degree Celsius for an hour.</p> |
− | <p>The number of the product: YBL8201005, YBL8201006, YBL8201007, YBL8201008, YBL8201009.</p> | + | <p>The number of the product: YBL8201005, YBL8201006, YBL8201007, YBL8201008, YBL8201009.</p> |
− | <p><strong>Recorder: Chenyang Li</strong> | + | <p><strong>Recorder: Chenyang Li</strong> |
− | <strong>PCR of pYeWGAP-BD</strong></p> | + | <strong>PCR of pYeWGAP-BD</strong></p> |
− | <p><strong>Procedure:</strong></p> | + | <p><strong>Procedure:</strong></p> |
− | <p>3.Prepare 4 PCR of pYeWGAP-BD tubes and sequentially add: | + | <p>3.Prepare 4 PCR of pYeWGAP-BD tubes and sequentially add: |
− | 4 μL Sterilized ddH<sub>2</sub>O, | + | 4 μL Sterilized ddH<sub>2</sub>O, |
− | + | 5 μL 2XHiFi Pfu, | |
− | + | 0.5 μL primer F, | |
− | + | 0.5 μL primer R, | |
− | 0.5 μL DNA template.</p> | + | 0.5 μL DNA template.</p> |
− | <p>4.PCR reaction | + | <p>4.PCR reaction |
− | Parameters setting:</p> | + | Parameters setting:</p> |
− | <table class="ui celled small table"> | + | <table class="ui celled small table"> |
− | <thead> | + | <thead> |
− | <tr> | + | <tr> |
− | + | <th>stage</th> | |
− | + | <th>temperature</th> | |
− | + | <th>time</th> | |
− | </tr> | + | </tr> |
− | </thead> | + | </thead> |
− | <tbody> | + | <tbody> |
− | <tr> | + | <tr> |
− | + | <td>step 1</td> | |
− | + | <td>95℃</td> | |
− | + | <td>10 min</td> | |
− | </tr> | + | </tr> |
− | <tr> | + | <tr> |
− | + | <td>step 2</td> | |
− | + | <td>95℃</td> | |
− | + | <td>30 s</td> | |
− | </tr> | + | </tr> |
− | <tr> | + | <tr> |
− | + | <td>step 3</td> | |
− | + | <td>58℃</td> | |
− | + | <td>45 s</td> | |
− | </tr> | + | </tr> |
− | <tr> | + | <tr> |
− | + | <td>step 4</td> | |
− | + | <td>72℃</td> | |
− | + | <td>45 s</td> | |
− | </tr> | + | </tr> |
− | <tr> | + | <tr> |
− | + | <td>step 5</td> | |
− | + | <td>72℃</td> | |
− | + | <td>10 min</td> | |
− | </tr> | + | </tr> |
− | <tr> | + | <tr> |
− | + | <td>step 6</td> | |
− | + | <td>4℃</td> | |
− | + | <td>--</td> | |
− | </tr> | + | </tr> |
− | </tbody> | + | </tbody> |
− | <table class="ui celled small table"> | + | <table class="ui celled small table"> |
− | <p>30 cycles(step 2 ~ step 4)</p> | + | <p>30 cycles(step 2 ~ step 4)</p> |
− | <p>results:</p> | + | <p>results:</p> |
− | <p><img src="https://static.igem.org/mediawiki/2016/thumb/6/65/T--USTC--NB_0820_3.jpeg/750px-T--USTC--NB_0820_3.jpeg" alt="a" /> | + | <p><img src="https://static.igem.org/mediawiki/2016/thumb/6/65/T--USTC--NB_0820_3.jpeg/750px-T--USTC--NB_0820_3.jpeg" alt="a" /> |
− | (from left to right: Trans 2K plus 2(contain Gelred),YBE8241001,1,YBE8241002,2,YBE8241003,3,YBE8241004,4,YeWGAP08171001,YeWGAPD8191002,BDD8191002,YBL8201005,Sterilized ddH<sub>2</sub>O) | + | (from left to right: Trans 2K plus 2(contain Gelred),YBE8241001,1,YBE8241002,2,YBE8241003,3,YBE8241004,4,YeWGAP08171001,YeWGAPD8191002,BDD8191002,YBL8201005,Sterilized ddH<sub>2</sub>O) |
− | </p> | + | </p> |
− | <h2>Date: 8.26</h2> | + | <h2>Date: 8.26</h2> |
− | <p><strong>Recorder: Yu Xie</strong> | + | <p><strong>Recorder: Yu Xie</strong> |
− | <strong>PCR of sfGFP1-10</strong></p> | + | <strong>PCR of sfGFP1-10</strong></p> |
− | <p><strong>Recorder: Yu Xie</strong> | + | <p><strong>Recorder: Yu Xie</strong> |
− | <strong>Double Digestion of sfGFP1-10</strong></p> | + | <strong>Double Digestion of sfGFP1-10</strong></p> |
− | <p><strong>Recorder: Yu Xie</strong> | + | <p><strong>Recorder: Yu Xie</strong> |
− | <strong>Double Digestion of pSB1C3</strong></p> | + | <strong>Double Digestion of pSB1C3</strong></p> |
− | <h2>Date: 8.27</h2> | + | <h2>Date: 8.27</h2> |
− | <p><strong>Recorder: Yu Xie</strong> | + | <p><strong>Recorder: Yu Xie</strong> |
− | <strong>Ligation of pSB1C3 and sfGFP1-10</strong></p> | + | <strong>Ligation of pSB1C3 and sfGFP1-10</strong></p> |
− | <p><strong>Recorder: Yu Xie</strong> | + | <p><strong>Recorder: Yu Xie</strong> |
− | <strong>Transformation of combinant plasmid pSB1C3 containing sfGFP1-10</strong></p> | + | <strong>Transformation of combinant plasmid pSB1C3 containing sfGFP1-10</strong></p> |
− | <p><strong>Recorder:Yu Xie</strong> | + | <p><strong>Recorder:Yu Xie</strong> |
− | <strong>Colony picking of combinant plasmid pSB1C3 containing sfGFP1-10</strong></p> | + | <strong>Colony picking of combinant plasmid pSB1C3 containing sfGFP1-10</strong></p> |
− | <p><strong>Recorder: Yu Xie</strong> | + | <p><strong>Recorder: Yu Xie</strong> |
− | <strong>PCR of sfGFP1-10</strong></p> | + | <strong>PCR of sfGFP1-10</strong></p> |
− | <p><strong>Recorder: Yu Xie</strong> | + | <p><strong>Recorder: Yu Xie</strong> |
− | <strong>Double Digestion of sfGFP1-10</strong></p> | + | <strong>Double Digestion of sfGFP1-10</strong></p> |
− | <p><strong>Plasmid Extraction for plasmid C3BD35</strong> | + | <p><strong>Plasmid Extraction for plasmid C3BD35</strong> |
− | <strong>Recorder: Xuefeng Meng</strong></p> | + | <strong>Recorder: Xuefeng Meng</strong></p> |
− | <p><strong>results</strong></p> | + | <p><strong>results</strong></p> |
− | <table class="ui celled small table"> | + | <table class="ui celled small table"> |
− | <thead> | + | <thead> |
− | <tr> | + | <tr> |
− | + | <th>sample</th> | |
− | + | <th>1</th> | |
− | + | <th>2</th> | |
− | + | <th>3</th> | |
− | </tr> | + | </tr> |
− | </thead> | + | </thead> |
− | <tbody> | + | <tbody> |
− | <tr> | + | <tr> |
− | + | <td>A260/A280</td> | |
− | + | <td>2.02</td> | |
− | + | <td>1.90</td> | |
− | + | <td>1.91</td> | |
− | </tr> | + | </tr> |
− | <tr> | + | <tr> |
− | + | <td>A260/A230</td> | |
− | + | <td>1.19</td> | |
− | + | <td>1.07</td> | |
− | + | <td>1.31</td> | |
− | </tr> | + | </tr> |
− | <tr> | + | <tr> |
− | + | <td>concentration(ng/μL)</td> | |
− | + | <td>17.0</td> | |
− | + | <td>15.5</td> | |
− | + | <td>27.9</td> | |
− | </tr> | + | </tr> |
− | </tbody> | + | </tbody> |
− | </table> | + | </table> |
− | <p><strong>PCR of sfGFP11</strong> | + | <p><strong>PCR of sfGFP11</strong> |
− | <strong>Recorder: Xuefeng Meng</strong> | + | <strong>Recorder: Xuefeng Meng</strong> |
− | <strong>results</strong> | + | <strong>results</strong> |
− | <img src="https://static.igem.org/mediawiki/2016/6/6a/T--USTC--NB_0826_1.jpeg" /> | + | <img src="https://static.igem.org/mediawiki/2016/6/6a/T--USTC--NB_0826_1.jpeg" /> |
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Latest revision as of 18:27, 19 October 2016
Notebook
Together we stand
Performers
DATE:8.24
Recorder: Yu Xie Plasmid Extraction for sfGFP11
Recorder: Yu Xie PCR of sup35
Procedure:
1.Prepare 8 PCR tubes and sequentially add:
sample | 1,2,3,4,5,6,7,8 | |||
---|---|---|---|---|
Sterilized ddH2O | 9 μL | |||
2×HiFi-PCR Master | 12.5 μL | |||
Template | 1.5 μL | |||
Primer 1 | 1 μL | |||
Primer 2 | 1 μL | |||
total | 25 μL |
stage | temperature | time | ||
---|---|---|---|---|
Pre-Duration | 95℃ | 10 min | ||
Duration | 95℃ | 1 min | ||
Anneal | 58℃ | 1 min | ||
Extend | 72℃ | 1 min 50 s | ||
Post-Extend | 72℃ | 10 min | ||
Final | 4℃ | -- |
sample | 1,2,3,4,5,6,7,8 | |||
---|---|---|---|---|
Sterilized ddH2O | 9 μL | |||
2×HiFi-PCR Master | 12.5 μL | |||
Template | 1.5 μL | |||
Primer 1 | 1 μL | |||
Primer 2 | 1 μL | |||
total | 25 μL |
stage | temperature | time | ||
---|---|---|---|---|
Pre-Duration | 95℃ | 10 min | ||
Duration | 95℃ | 1 min | ||
Anneal | 58℃ | 1 min | ||
Extend | 72℃ | 1 min 50s | ||
Post-Extend | 72℃ | 10 min | ||
Final | 4℃ | -- |
stage | temperature | time | ||
---|---|---|---|---|
step 1 | 95℃ | 10 min | ||
step 2 | 95℃ | 30 s | ||
step 3 | 58℃ | 45 s | ||
step 4 | 72℃ | 45 s | ||
step 5 | 72℃ | 10 min | ||
step 6 | 4℃ | -- |
sample | YBE8241001 | YBE8241002 | YBE8241003 | YBE8241004 |
---|---|---|---|---|
A260/A280 | 1.88 | 1.86 | 1.89 | 1.88 |
A260/A230 | 2.27 | 1.92 | 2.22 | 2.22 |
concentration(ng/μL) | 203.1 | 174.7 | 181.3 | 177.8 |
stage | temperature | time | |
---|---|---|---|
step 1 | 95℃ | 10 min | |
step 2 | 95℃ | 30 s | |
step 3 | 58℃ | 45 s | |
step 4 | 72℃ | 45 s | |
step 5 | 72℃ | 10 min | |
step 6 | 4℃ | -- |
sample | 1,2,3,4,5,6,7,8 | ||
---|---|---|---|
Sterilized ddH2O | 9 μL | ||
2×HiFi-PCR Master | 12.5 μL | ||
Template | 1.5 μL | ||
Primer 1 | 1 μL | ||
Primer 2 | 1 μL | ||
total | 25 μL |
stage | temperature | time | |
---|---|---|---|
Pre-Duration | 95℃ | 10 min | |
Duration | 95℃ | 1 min | |
Anneal | 58℃ | 1 min | |
Extend | 72℃ | 1 min 50 s | |
Post-Extend | 72℃ | 10 min | |
Final | 4℃ | -- |
stage | temperature | time | |
---|---|---|---|
step 1 | 95℃ | 10 min | |
step 2 | 95℃ | 30 s | |
step 3 | 58℃ | 45 s | |
step 4 | 72℃ | 45 s | |
step 5 | 72℃ | 10 min | |
step 6 | 4℃ | -- |
sample | 1 | 2 | 3 |
---|---|---|---|
A260/A280 | 2.02 | 1.90 | 1.91 |
A260/A230 | 1.19 | 1.07 | 1.31 |
concentration(ng/μL) | 17.0 | 15.5 | 27.9 |
PCR of sfGFP11 Recorder: Xuefeng Meng results
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