Team:Hong Kong CUHK/protocol

From 2013.igem.org

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</div>
</div>
<div id="middle">
<div id="middle">
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<div id="containerX2" style="margin: 0px auto;">
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<div id="containerX2" align="centre">
<div id="heading" style="margin: 0px auto;"><h2>Project Overview</h2></div>
<div id="heading" style="margin: 0px auto;"><h2>Project Overview</h2></div>
<div id="partable">
<div id="partable">
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               2 μl         10X NEB  Buffer2<br />
               2 μl         10X NEB  Buffer2<br />
               2 μl         10X BSA<br />
               2 μl         10X BSA<br />
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       0.5 μl       Enzyme 1<br />
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               0.5 μl       Enzyme 1<br />
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         0.5 μl       Enzyme 2<br />
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               0.5 μl       Enzyme 2<br />
       # At least 200ng DNA should be  added<br />
       # At least 200ng DNA should be  added<br />
       * Water is added first and the  template the last<br />
       * Water is added first and the  template the last<br />
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       <p>1. Mix the components as  follows to prepare a 10 μl reaction mixture:<br />
       <p>1. Mix the components as  follows to prepare a 10 μl reaction mixture:<br />
               0.5 μl         Water<br />
               0.5 μl         Water<br />
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                    1 μl        10X  ligation buffer<br />
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              1 μl        10X  ligation buffer<br />
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                0.5 μl        T4 DNA  ligase<br />
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               0.5 μl        T4 DNA  ligase<br />
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                  2 μl        Vector<br />
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               2 μl        Vector<br />
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                  6 μl        Insert<br />
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               6 μl        Insert<br />
         2. Incubate the reactions at  16oC overnight, 22oC for 1 h or stand in RT for 10min.</p>
         2. Incubate the reactions at  16oC overnight, 22oC for 1 h or stand in RT for 10min.</p>
     </p2>
     </p2>
     </p>
     </p>
     <p><a href="#top">Back To Top</a></p>
     <p><a href="#top">Back To Top</a></p>
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    <p><a name="a2.1" id="a2.1"></a>
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 +
<p><a name="a2.1" id="a2.1"></a>
     <h4><strong>2.1 Cell Viability Test with Voltage applied</strong></h4>
     <h4><strong>2.1 Cell Viability Test with Voltage applied</strong></h4>
<p2>
<p2>
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<ul>
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<p>We are applying continuous DC Voltage to  the samples by using batteries.</p>
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  <li>Experimental Groups</li>
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  <ul>
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</ul>
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    <li>Cells used: BL21 transformed with 2µL PSB1C3 + RFP</li>
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<p>            Cells used: BL21 transformed with 2µL PSB1C3 + RFP
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   </ul>
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   Spread  on Chloramphenicol agar plate and incubate overnight in 37 °C <br />
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  <p>        Spread  on Chloramphenicol agar plate and incubate overnight in 37 °C <br />
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</p>
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  </p>
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<ul>
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  <ul>
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  <li>Control group</li>
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    <li>Pick 8 clones  into tubes with 10 mL of LB solution.   Add 10 µL of Chloramphenicol to experimental group tubes. Do  overnight shake in incubator for 16 hours to make the cells saturated.</li>
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</ul>
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  </ul>
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<p>            Cells used: BL21 transformed with 2µL water.
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  <ul>
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   Spread on Blank agar plate and incubate overnight in 37 °C </p>
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    <li>After 16  hours, take out the tubes from the incubator. </li>
 +
  </ul>
 +
  <p>&nbsp;</p>
 +
  <ul>
 +
    <li>Normalize the samples to around 400 – 600 ABS with OD check (using 600nm wavelength) by  adding extra LB + Chloramphenicol.</li>
 +
   </ul>
 +
  <p>        </p>
 +
  <ul>
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    <li>Place 5 mL of  sample to each experimental tube. (6 voltages x 3 = 18 tubes)</li>
 +
  </ul>
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  <ul>
 +
    <li>Next, we  perform the experiment with voltage applied to each tubes.  We use one 9V battery for 9V, two 9V batteries  in series for 18 V, three 9V batteries in series for 27 V, four 9V batteries in  series for 36 V, and five 9V batteries in series for 45 V. Tubes will be shaken  under 250 rpm and 25°C for 24 hours. For each voltage, we have three samples.</li>
 +
  </ul>
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  <p align="center"><img width="268" height="183" src="https://static.igem.org/mediawiki/2013/b/be/%2B9V.jpg" alt="Macintosh HD:Users:melisajunata:Desktop:+9V.jpg" /><img width="276" height="286" src="https://static.igem.org/mediawiki/2013/8/87/%2B18V.jpg" alt="Macintosh HD:Users:melisajunata:Desktop:+18V.jpg" /> <br />
 +
    Fig.  1 +9V circuit                   Fig. 2  +18V circuit</p>
 +
  <p>&nbsp;</p>
 +
  <p>&nbsp;</p>
 +
  <p align="center"><img width="276" height="362" src="https://static.igem.org/mediawiki/2013/8/81/%2B27V.jpg" alt="Macintosh HD:Users:melisajunata:Desktop:+27.jpg" /><img width="272" height="376" src="https://static.igem.org/mediawiki/2013/2/2a/%2B36V.jpg" alt="Macintosh HD:Users:melisajunata:Documents:iGEM:1WC:Wiki and presentation:36 V.jpg" /> <br />
 +
    Fig. 3  +27V circuit                         Fig. 4 +36V circuit<br />
 +
</p> <p align="center"><img width="264" height="409" src="https://static.igem.org/mediawiki/2013/8/88/%2B45V.jpg" alt="Macintosh HD:Users:melisajunata:Documents:iGEM:1WC:Wiki and presentation:45 V.png" /> <br />
 +
    Fig 5. +45V circuit</p>
 +
  <ul>
 +
    <li>After 24  hours, we take the tubes out. Then we take 5 µL of each sample to perform serial  dilution. We performed 10X, 100X, 1000X and 10,000X dilution.</li>
 +
  </ul>
 +
  <p>&nbsp;</p>
 +
  <ul>
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    <li>Take 1 µL  from each diluted solution to the Chloramphenicol agar &lsquo;plate  table&rsquo; and streak on the square carefully. Then incubate the plates for 12  hours in 37 °C.</li>
 +
  </ul>
 +
  <p align="center">The &lsquo;Plate Table&rsquo;<br />
 +
    <img width="553" height="414" src="https://static.igem.org/mediawiki/2013/a/a3/Plate.jpg" alt="Macintosh HD:Users:melisajunata:Desktop:picts result:IMG_2676.JPG" /></p></p2>
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<p>           1)Pick 4 clones from each group into  tubes with 10 mL of LB solution. Add 10 µL of Chloramphenicol to experimental group tubes. Do overnight shake in incubator for 12 hours to make the cells saturated.After 12 hours, take out the tubes  from the incubator. </p>
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<p><a href="#top">Back To Top</a></p>
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<p>2)Prepare the following tubes for each  control and experimental  groups.</p>
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<p><a name="a2.2" id="a2.2"></a>
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<table border="1" cellspacing="0" cellpadding="0" width="624">
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  <tr>
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    <td width="92" valign="top"><p>Test    Set</p></td>
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    <td width="70" valign="top"><p>27    V</p></td>
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    <td width="70" valign="top"><p>18    V</p></td>
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    <td width="70" valign="top"><p>9 V</p></td>
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    <td width="70" valign="top"><p>-9V</p></td>
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    <td width="71" valign="top"><p>-18V</p></td>
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    <td width="71" valign="top"><p>-27V</p></td>
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    <td width="109" valign="top"><p>OV(    control)</p></td>
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  </tr>
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  <tr>
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    <td width="92" valign="top"><p>1</p></td>
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    <td width="70" valign="top"><p>0.8    mL</p></td>
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    <td width="70" valign="top"><p>0.8    mL</p></td>
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    <td width="70" valign="top"><p>0.8    mL</p></td>
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    <td width="70" valign="top"><p>0.8    mL</p></td>
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    <td width="71" valign="top"><p>0.8    mL</p></td>
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    <td width="71" valign="top"><p>0.8    mL</p></td>
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    <td width="109" valign="top"><p>0.8    mL</p></td>
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  </tr>
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  <tr>
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    <td width="92" valign="top"><p>2</p></td>
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    <td width="70" valign="top"><p>0.8    mL</p></td>
+
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    <td width="70" valign="top"><p>0.8    mL</p></td>
+
-
    <td width="70" valign="top"><p>0.8    mL</p></td>
+
-
    <td width="70" valign="top"><p>0.8    mL</p></td>
+
-
    <td width="71" valign="top"><p>0.8    mL</p></td>
+
-
    <td width="71" valign="top"><p>0.8    mL</p></td>
+
-
    <td width="109" valign="top"><p>0.8    mL</p></td>
+
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  </tr>
+
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  <tr>
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    <td width="92" valign="top"><p>3</p></td>
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    <td width="70" valign="top"><p>0.8    mL</p></td>
+
-
    <td width="70" valign="top"><p>0.8    mL</p></td>
+
-
    <td width="70" valign="top"><p>0.8    mL</p></td>
+
-
    <td width="70" valign="top"><p>0.8    mL</p></td>
+
-
    <td width="71" valign="top"><p>0.8    mL</p></td>
+
-
    <td width="71" valign="top"><p>0.8    mL</p></td>
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    <td width="109" valign="top"><p>0.8    mL</p></td>
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-
  </tr>
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-
</table>
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<p>Then, we add 3.2 mL of LB and 4 µL  of Chloramphenicol (35 mg/L) to each  of the experimental group tubes. For control group, we only add 3.2 mL of LB. </p>
+
-
<br></br><br></br><br></br><br></br><br></br><br></br><br></br><br></br><br></br><br></br><br></br><br></br><br></br><br></br><br></br><br></br><br></br><br></br><br></br><br></br><br></br><br></br><br></br><br></br>
+
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3)Before connecting the voltage to  each sample, we take 0.8 mL of each sample to test for initial Optical Density measurement  using Spectrophotometry.
+
-
<p>4)Next, we perform the experiment  with voltage applied to each tubes.  We use  one 9V battery for 9V, two 9V batteries in series for 18 V and three 9V  batteries in series for 27 V. Tubes will be shaken under 250 rpm and 25°C for  16 hours. </p></p2>
+
<h4><strong>2.2 Cell Viability Test with BaP</strong></h4>
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<div id="circuitcontainer">
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<div id="Cone" >
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  <p><img src="https://static.igem.org/mediawiki/igem.org/c/c5/Oneplusnine.jpg" width="361" height="260" /></p>
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  <p>
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    <p2>Fig. 1 +9V circuit</p2>
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  </p>
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</div>
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<div id="Ctwo">
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  <p><img src="https://static.igem.org/mediawiki/igem.org/e/eb/Twominusnine.jpg" width="330" height="242" /></p>
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  <p>
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    <p2> Fig. 2 -9V  circuit</p2>
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  </p>
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</div>
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<div id="Cfive">
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  <p><img src="https://static.igem.org/mediawiki/igem.org/3/30/Fiveplustwosev.jpg" width="322" height="424" /></p>
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  <p2>Fig. 5 +27V circuit</p2>
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</div>
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<div id="Cfour">
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  <p><img src="https://static.igem.org/mediawiki/igem.org/6/6c/Fourminuseteen.jpg" width="318" height="347" /></p>
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  <p>
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    <p2>Fig. 4 -18V circuit</p2>
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-
  </p>
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</div>
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<div id="Cthree">
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  <p><img src="https://static.igem.org/mediawiki/igem.org/5/59/Threepluseteen.jpg" width="322" height="335" /></p>
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  <p>
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    <p2>Fig. 3 +18V circuit</p2>
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  </p>
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</div>
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<div id="Csix">
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  <p><img src="https://static.igem.org/mediawiki/igem.org/e/e2/Sixminustwosev.jpg" width="341" height="439" /></p>
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  <p2>Fig.  6 -27V circuit</p2>
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</div>
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</div>
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<div id="partable2">
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<p2>
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<p><br></br><br></br><br></br><br></br><br></br><br></br><br></br><br></br><br></br><br></br><br></br><br></br><br></br><br></br><br></br><br></br><br></br><br></br><br></br><br></br>5)After 16 hours, we take the tubes  out. Then we take 0.8 mL of each sample to perform final Optical Density  measurement using Spectrophotometry.</p>
+
-
<p>6)Calculate Changes in Optical  Density= Final Optical Density – Initial Optical Density</p>
+
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<p>7)Plot the graph for three test  sets. </p>
+
-
<p><a href="#top">Back To Top</a></p>
+
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<p><a name="a2.2" id="a2.2"></a><h4><strong>2.2 Cell Viability Test with BaP</strong></h4>
+
<p2>
<p2>
     <ol>
     <ol>

Revision as of 04:59, 27 October 2013

iGEM CUHK