Difference between revisions of "Team:SEU/Experiments"

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<h1>Experiments</h1>
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<p>Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.</p>
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Please remember to put all characterization and measurement data for your parts on the corresponding Registry part pages.  
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                                          <h2>Experiments</h2>
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                                          <p>To demonstrate our calculations simulated by computer, we carried out following experiments. We will show you these experiments in chronological order and let you experience our trial and error and improvements in experimental method.</p>
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                                          <h4>All the experiments can be categorized into six parts:</h4>
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                                          <p>a. Acquire single chain DNA sequences used in wet experiments (we got these sequences by our home-made software tool)<br>
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                                          b.  Synthesize DNA in part 1 (in fact we bought them from Sangon Biotech)<br>
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                                          c.  Dissolve, dilute, mix<br>
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                                          d.  Denature and anneal (acquire the results of fluorescence intensity by qRT-PCR simultaneously)<br>
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                                          e.  Purify and quantify the reactant complexes<br>
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                                          f. Characterization via PAGE</p>
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                                          <h4>Basic experiment details are as follows:</h4>
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                                          <h5>1. Dissolve, dilute, mix</h5>
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                                          <p>All the single chain DNAs were dissolved in DEPC-treated water to form 100 M solutions respectively and saved at 4 ℃.<br>
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                                          Reactant complexes were annealed together at 20 uM in Tris-acetate-EDTA buffer containing 12.5 mM Mg2+(1×TAE/Mg2+) and saved at 4 ℃ for further experiments.</p>
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                                          <h5>2. Gel electrophoresis</h5>
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                                          <p>To examine the products of every kinetics experiments and purify the reactant complexes, 12% non-denaturing PAGE was run at 40 V for about 3.5 hours. After staining with GelRed (Biotium, 1:10000) for 30 min, the gel was scanned on a gel imaging system (Tanon 3500R).<br>
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                                          Formulation for 12% native PAGE is as follows:
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                                            <table border="1">
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                                              <td>29:1 30% Acrylamide/bis</td>
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                                              <td>Deionized Water</td>
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                                              <td>5xTBE Buffer</td>
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                                              <td>10% APS</td>
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                                              <td>TEMED</td>
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                                              <td>Total Volume</td>
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                                            </tr> 
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                                              <td>4mL</td>
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                                              <td>3.93mL</td>
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                                              <td>2mL</td>
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                                              <td>0.07mL</td>
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                                              <td>0.007mL</td>
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                                              <td>10mL</td>
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                                            </tr>
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<h3>What should this page contain?</h3>
 
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<li> Protocols </li>
 
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<li> Experiments </li>
 
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<li> Documentation of the development of your project </li>
 
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<h3>Inspiration</h3>
 
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<li><a href="https://2014.igem.org/Team:Colombia/Protocols">2014 Colombia </a></li>
 
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<li><a href="https://2014.igem.org/Team:Imperial/Protocols">2014 Imperial </a></li>
 
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<li><a href="https://2014.igem.org/Team:Caltech/Project/Experiments">2014 Caltech </a></li>
 
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Revision as of 07:56, 10 October 2019





Experiments

To demonstrate our calculations simulated by computer, we carried out following experiments. We will show you these experiments in chronological order and let you experience our trial and error and improvements in experimental method.

All the experiments can be categorized into six parts:

a. Acquire single chain DNA sequences used in wet experiments (we got these sequences by our home-made software tool)
b. Synthesize DNA in part 1 (in fact we bought them from Sangon Biotech)
c. Dissolve, dilute, mix
d. Denature and anneal (acquire the results of fluorescence intensity by qRT-PCR simultaneously)
e. Purify and quantify the reactant complexes
f. Characterization via PAGE

Basic experiment details are as follows:

1. Dissolve, dilute, mix

All the single chain DNAs were dissolved in DEPC-treated water to form 100 M solutions respectively and saved at 4 ℃.
Reactant complexes were annealed together at 20 uM in Tris-acetate-EDTA buffer containing 12.5 mM Mg2+(1×TAE/Mg2+) and saved at 4 ℃ for further experiments.

2. Gel electrophoresis

To examine the products of every kinetics experiments and purify the reactant complexes, 12% non-denaturing PAGE was run at 40 V for about 3.5 hours. After staining with GelRed (Biotium, 1:10000) for 30 min, the gel was scanned on a gel imaging system (Tanon 3500R).
Formulation for 12% native PAGE is as follows:

29:1 30% Acrylamide/bis Deionized Water 5xTBE Buffer 10% APS TEMED Total Volume
4mL 3.93mL 2mL 0.07mL 0.007mL 10mL