Difference between revisions of "Team:IIT Kharagpur/Notebook"

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<script src="https://2015.igem.org/Team:IIT_Kharagpur/bootstrapjs?action=raw&amp;ctype=text/javascript"></script>
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</head>
 
</head>
 
<body>
 
<body>
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<div class="container">
 
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<div class="jumbotron">
 
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<h1>Notebook Page</h1>
 
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<p><h2 class="heading">(Log of day-to-day work done)</h2></p>
 
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</div>
 
  
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<p><h2 class="heading"><u class="linecolor">WEEK-1( July 6 - July 10 )</u></h2></p>
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<p>
 
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<h3>6th July 2015</h3>
 
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<p>
 
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<ul>
 
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<li>LB media prepared (500 ml), autoclaved and stored at 4 degrees.</li>
 
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<li>250 ml of LB agar prepared and poured into the plates (chloramphenicol antibiotic
 
  
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used- concentration 1 &micro;l/ml).</li>
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<script type="text/javascript">
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</ul>
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$(window).scroll(function() {   
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</p>
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    var scroll1 = $(window).scrollTop();
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</p>
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<h3>7th July 2015</h3>
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<p>
+
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<ul>
+
−
<li>DNA from wells dissolved into 10 &micro;l distilled water.<u> DNA kit plate instructions</u></li>
+
−
<li>Transformed 4&micro;l of DNA into 100&micro;l of E.Coli DH5&alpha;.<u> transformation procedure</u></li>
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<li>100&micro;l of transformed cells were plated on chloramphenicol resistant plates.and left
+
  
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overnight to grow.</li>
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        $(".menu").addClass("menu-show");
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        $(".menu").removeClass("menu-show");
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<h3>8th July 2015</h3>
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    }
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<p>
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});
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<ul>
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        </script>
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<li>Colonies for luxPR promoter (BBa_K546003) constructs were observed. A colony
+
 +
 +
<div class="jumbotron jumbo-home">
 +
  
−
was picked and inoculated in 3 ml culture for glycerol stock preparation.</li>
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<div class="menu">
−
<li>No colony observed for luxR protein (BBa_R6002) construct.</li>
+
        <ul>
−
</ul>
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          <li class="menu-logo"><a href="https://2015.igem.org/Team:IIT_Kharagpur"><img class='menu-logo-img' src='https://static.igem.org/mediawiki/2015/5/59/IIT_Kharagpur_menu_team_logo.png'/><div class="menu-logo-text">IGEM IIT </br> Kharagpur</div></a></li>
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</p>
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          <li class="menu-li"><a href="https://2015.igem.org/Team:IIT_Kharagpur"><img class='menu-img' src='https://static.igem.org/mediawiki/2015/6/66/IIT_Kharagpur_menu_home.png'/><div class="menu-text">Home</div></a></li>
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</p>
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          <li class="menu-li"><a href="https://2015.igem.org/Team:IIT_Kharagpur/project"><img class="menu-project-img menu-img" src="https://static.igem.org/mediawiki/2015/6/61/IIT_Kharagpur_menu_project.png" /><div class="menu-text">Project</div></a>
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<h3>9th July 2015</h3>
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          <ul>
−
<p>
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                  <li><a href="https://2015.igem.org/Team:IIT_Kharagpur/project">About</li>
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<ul>
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                  <li><a href="https://2015.igem.org/Team:IIT_Kharagpur/parts">Parts</li>
−
<li>Transformation repeated for luxR protein construct. <u>transformation procedure</u></li>
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                  <li><a href="https://2015.igem.org/Team:IIT_Kharagpur/modeling">Modeling</a></li>
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<li>Culture prepared for plasmid isolation for luxPR promoter. Colony was picked and
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            </ul>
 +
          </li>
 +
          <li class="menu-li"><a href="https://2015.igem.org/Team:IIT_Kharagpur/team"><img class="menu-team-img menu-img" src="https://static.igem.org/mediawiki/2015/2/2d/IIT_Kharagpur_menu_team.png" /><div class="menu-text">Team</div></a></li>
 +
          <li class="menu-li"><a href="https://2015.igem.org/Team:IIT_Kharagpur/notebook"><img class="menu-notebook-img menu-img" src="https://static.igem.org/mediawiki/2015/f/f6/IIT_Kharagpur_menu_notebook.png" /><div class="menu-text">Notebook</div></a></li>
 +
          <li class="menu-li"><a href="https://2015.igem.org/Team:IIT_Kharagpur/practices"><img class="menu-modeling-img menu-img" src="https://static.igem.org/mediawiki/2015/7/7e/IIT_Kharagpur_menu_modeling.png" /><div class="menu-text">Practices</div></a>
 +
               
 +
          </li>
 +
         
 +
          <li class="menu-li"><a href="https://2015.igem.org/Team:IIT_Kharagpur/safety"><img class="menu-achievements-img menu-img" src="https://static.igem.org/mediawiki/2015/2/2f/IIT_Kharagpur_menu_safety.png" /><div class="menu-text">Safety</div></a></li>
 +
         
 +
          <li class="menu-li"><a href="https://2015.igem.org/Team:IIT_Kharagpur/blog.html"><img class="menu-safety-img menu-img" src="https://static.igem.org/mediawiki/2015/2/27/IIT_Kharagpur_menu_blog.png" /><div class="menu-text">Blog</div></a></li>
 +
        </ul>
  
−
inoculated in 10 ml culture.</li>
+
      </div>
−
</ul>
+
−
</p>
+
−
</p>
+
−
<h3>10th July 2015</h3>
+
−
<p>
+
−
<ul>
+
−
<li>No colony observed for luxR protein construct.</li>
+
−
<li>Plasmid isolation done for luxPR promoter.</li>
+
−
<li>Gel electrophoresis done for luxPR promoter.
+
−
                <p><img src="https://static.igem.org/mediawiki/2015/b/b0/Team_IIT_Kharagpur_luxPR_promoter.jpg" class="img-responsive"></p>
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                </li>
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+
−
<li>Lane 7 : 1kb marker<br>
+
  
−
            Lane 8 : luxPR promoter</li>
+
<div class="notebook-text-div1">
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</ul>
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<img class="notebook-text1"src="img/notebook-text.png" />
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</p>
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</div>
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</p>
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<div class="notebook-text-div2">
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<a  href="#"><img class="notebook-text2"src="img/notebook-icon.png" /></a>
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</div>
 +
 +
 +
</div>
  
  
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</div>
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−
<!-- footer start -->
+
<div class="color-rail">
−
  <footer class="footer">
+
<div class="white"></div>
−
<div class="container">
+
<div class="light-gray"></div>
−
<p>Copyright © 2015 iGEM Team IIT Kharagpur</p>
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<div class="gray"></div>
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<div class="dark-gray"></div>
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<div class="black"></div>
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<div class="dark-teal"></div>
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<div class="teal"></div>
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<div class="orange"></div>
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</div>
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<div class="container notebook-mid">
 +
<div class="row">
 +
<div class="col-lg-3 col-md-3">
 +
<div class="notebook-side" hidden-xs hidden-sm hidden-md data-spy="affix" data-offset-top="800">
 +
<div class="calendar">
  
 +
<header>
 +
 +
<h3>July</h3>
 +
 +
</header>
 +
 +
<table>
 +
 +
<thead>
 +
 +
<tr>
 +
 +
<td>Mo</td>
 +
<td>Tu</td>
 +
<td>We</td>
 +
<td>Th</td>
 +
<td>Fr</td>
 +
<td>Sa</td>
 +
<td>Su</td>
 +
 +
</tr>
 +
 +
</thead>
 +
 +
<tbody>
 +
 +
<tr>
 +
<td class="prev-month">29</td>
 +
<td class="prev-month">30</td>
 +
<td class="">1</td>
 +
<td class="">2</td>
 +
<td class="">3</td>
 +
<td class="">4</td>
 +
<td>5</td>
 +
</tr>
 +
<tr>
 +
<td class="event"><a data-scroll href="#6-july"><div>6</div></a></td>
 +
<td class="event"><a data-scroll href="#7-july"><div>7</div></a></td>
 +
<td class="event"><a data-scroll href="#8-july"><div>8</div></a></td>
 +
<td class="event"><a data-scroll href="#9-july"><div>9</div></a></td>
 +
<td class="event"><a data-scroll href="#10-july"><div>10</div></a></td>
 +
<td>11</td>
 +
<td>12</td>
 +
</tr>
 +
<tr>
 +
<td>13</td>
 +
<td>14</td>
 +
<td>15</td>
 +
<td>16</td>
 +
<td>17</td>
 +
<td>18</td>
 +
<td>19</td>
 +
</tr>
 +
<tr>
 +
<td>20</td>
 +
<td class="event"><a data-scroll href="#21-july"><div>21</div></a></td>
 +
<td class="event"><a data-scroll href="#22-july"><div>22</div></a></td>
 +
<td class="event"><a data-scroll href="#23-july"><div>23</div></a></td>
 +
<td class="event"><a data-scroll href="#24-july"><div>24</div></a></td>
 +
<td>25</td>
 +
<td class="event"><a data-scroll href="#26-july"><div>26</td>
 +
</tr>
 +
 +
<tr>
 +
<td>27</td>
 +
<td class="event"><a data-scroll href="#28-july"><div>28</div></a></td>
 +
<td class="event"><a data-scroll href="#29-july"><div>29</div></a></td>
 +
<td class="event"><a data-scroll href="#30-july"><div>30</div></a></td>
 +
<td class="event"><a data-scroll href="#31-july"><div>31</div></a></td>
 +
<td class="next-month">1</td>
 +
<td class="next-month">2</td>
 +
</tr>
 +
<tr>
 +
<td class="next-month">3</td>
 +
<td class="next-month">4</td>
 +
<td class="next-month">5</td>
 +
<td class="next-month">6</td>
 +
<td class="next-month">7</td>
 +
<td class="next-month">8</td>
 +
<td class="next-month">9</td>
 +
</tr>
 +
 +
</tbody>
 +
 +
</table>
 +
 +
<header>
 +
 +
<h3>August</h3>
 +
 +
</header>
 +
 +
<table>
 +
 +
<thead>
 +
 +
<tr>
 +
 +
<td>Mo</td>
 +
<td>Tu</td>
 +
<td>We</td>
 +
<td>Th</td>
 +
<td>Fr</td>
 +
<td>Sa</td>
 +
<td>Su</td>
 +
 +
</tr>
 +
 +
</thead>
 +
 +
<tbody>
 +
 +
<tr>
 +
<td class="prev-month">27</td>
 +
<td class="prev-month">28</td>
 +
<td class="prev-month">29</td>
 +
<td class="prev-month">30</td>
 +
<td class="prev-month">31</td>
 +
<td class="event"><a data-scroll href="#1-august"><div>1</div></a></td>
 +
<td>2</td>
 +
</tr>
 +
<tr>
 +
<td>3</td>
 +
<td>4</td>
 +
<td>5</td>
 +
<td>6</td>
 +
<td>7</td>
 +
<td>8</td>
 +
<td>9</td>
 +
</tr>
 +
<tr>
 +
<td>10</td>
 +
<td>11</td>
 +
<td>12</td>
 +
<td>13</td>
 +
<td>14</td>
 +
<td>15</td>
 +
<td>16</td>
 +
</tr>
 +
<tr>
 +
<td>17</td>
 +
<td>18</td>
 +
<td>19</td>
 +
<td>20</td>
 +
<td>21</td>
 +
<td>22</td>
 +
<td>23</td>
 +
</tr>
 +
 +
<tr>
 +
<td>24</td>
 +
<td>25</td>
 +
<td>26</td>
 +
<td>27</td>
 +
<td>28</td>
 +
<td>29</td>
 +
<td>30</td>
 +
</tr>
 +
<tr>
 +
<td>31</td>
 +
<td class="next-month">1</td>
 +
<td class="next-month">2</td>
 +
<td class="next-month">3</td>
 +
<td class="next-month">4</td>
 +
<td class="next-month">5</td>
 +
<td class="next-month">6</td>
 +
</tr>
 +
 +
</tbody>
 +
 +
</table>
 +
 +
<header>
 +
 +
<h3>September</h3>
 +
 +
</header>
 +
 +
<table>
 +
 +
<thead>
 +
 +
<tr>
 +
 +
<td>Mo</td>
 +
<td>Tu</td>
 +
<td>We</td>
 +
<td>Th</td>
 +
<td>Fr</td>
 +
<td>Sa</td>
 +
<td>Su</td>
 +
 +
</tr>
 +
 +
</thead>
 +
 +
<tbody>
 +
 +
<tr>
 +
<td class="prev-month">31</td>
 +
<td class="prev-month">1</td>
 +
<td class="prev-month">2</td>
 +
<td class="prev-month">3</td>
 +
<td class="prev-month">4</td>
 +
<td class="prev-month">5</td>
 +
<td>6</td>
 +
</tr>
 +
<tr>
 +
<td>7</td>
 +
<td>8</td>
 +
<td>9</td>
 +
<td>10</td>
 +
<td>11</td>
 +
<td>12</td>
 +
<td>13</td>
 +
</tr>
 +
<tr>
 +
<td>14</td>
 +
<td>15</td>
 +
<td>16</td>
 +
<td>17</td>
 +
<td>18</td>
 +
<td>19</td>
 +
<td>20</td>
 +
</tr>
 +
<tr>
 +
<td>21</td>
 +
<td>22</td>
 +
<td>23</td>
 +
<td>24</td>
 +
<td>25</td>
 +
<td>26</td>
 +
<td>27</td>
 +
</tr>
 +
 +
<tr>
 +
<td>28</td>
 +
<td>29</td>
 +
<td>30</td>
 +
<td class="next-month">1</td>
 +
<td class="next-month">2</td>
 +
<td class="next-month">3</td>
 +
<td class="next-month">4</td>
 +
 +
</tr>
 +
<tr>
 +
<td class="next-month">5</td>
 +
<td class="next-month">6</td>
 +
<td class="next-month">7</td>
 +
<td class="next-month">8</td>
 +
<td class="next-month">9</td>
 +
<td class="next-month">10</td>
 +
<td class="next-month">11</td>
 +
</tr>
 +
 +
</tbody>
 +
 +
</table>
 +
 +
</div> <!-- end calendar -->
 +
</div>
 
</div>
 
</div>
  
−
</footer>
+
<div class="col-lg-9 col-md-9 col-sm-12">
−
<!-- footer end -->
+
<div class="notebook-data-box1">
 +
<h2> Week One ( July 6 - July 10 )</h2>
 +
<div class="panel panel-success">
 +
  <div class="panel-heading">
 +
    <h3 class="panel-title" id="6-july">6th July 2015</h3>
 +
  </div>
 +
 
 +
 
 +
  <ul class="list-group">
 +
    <li class="list-group-item">LB media prepared (500 ml), autoclaved and stored at 4 degrees.</li>
 +
    <li class="list-group-item">250 ml of LB agar prepared and poured into the plates (chloramphenicol antibiotic used- concentration 1 micro litre/ml)</li>
 +
  </ul>
 +
</div>
 +
 
 +
<div class="panel panel-success">
 +
  <div class="panel-heading">
 +
    <h3 class="panel-title" id="7-july">7th July 2015</h3>
 +
  </div>
 +
 
 +
 
 +
  <ul class="list-group">
 +
    <li class="list-group-item">DNA from wells dissolved into 10 μl distilled water. DNA kit plate instructions</li>
 +
    <li class="list-group-item">Transformed 4μl of DNA into 100μl of E.Coli DH5ɑ. transformation procedure)</li>
 +
    <li class="list-group-item">100μl of transformed cells were plated on chloramphenicol resistant plates.and left overnight to grow.</li>
 +
  </ul>
 +
</div>
 +
 
 +
<div class="panel panel-success">
 +
  <div class="panel-heading">
 +
    <h3 class="panel-title" id="8-july">8th July 2015</h3>
 +
  </div>
 +
 
 +
 
 +
  <ul class="list-group">
 +
    <li class="list-group-item">Colonies for luxPR promoter (BBa_R6002) constructs were observed. A colony was picked and inoculated in 3 ml culture for glycerol stock preparation.</li>
 +
    <li class="list-group-item">No colony observed for luxR protein (BBa_K546003) construct.</li>
 +
  </ul>
 +
</div>
 +
 
 +
<div class="panel panel-success">
 +
  <div class="panel-heading">
 +
    <h3 class="panel-title" id="9-july">9th July 2015</h3>
 +
  </div>
 +
 
 +
 
 +
  <ul class="list-group">
 +
    <li class="list-group-item">Transformation repeated for luxR protein construct  .transformation procedure</li>
 +
    <li class="list-group-item">Culture prepared for plasmid isolation for luxPR promoter. Colony was picked and inoculated in 10 ml culture.</li>
 +
  </ul>
 +
</div>
 +
 
 +
<div class="panel panel-success">
 +
  <div class="panel-heading">
 +
    <h3 class="panel-title" id="10-july">10th July 2015</h3>
 +
  </div>
 +
 
 +
  <ul class="list-group">
 +
    <li class="list-group-item">No colony observed for luxR protein construct</li>
 +
    <li class="list-group-item">Plasmid isolation done for luxPR promoter</li>
 +
    <li class="list-group-item">Gel electrophoresis done for luxPR promoter</li>
 +
  </ul>
 +
 
 +
  <div class="panel-body">
 +
    <img class="img-responsive" src="img/image00.jpg">
 +
  </div>
 +
 
 +
  <ul class="list-group">
 +
    <li class="list-group-item well">Lane 7 : 1kb marker </br> Lane 8 : luxPR promoter</li>
 +
    <li class="list-group-item">Gel electrophoresis done for luxPR promoter</li>
 +
  </ul>
 +
 
 +
  <table class="table" id="week-one">
 +
    <thead>
 +
 +
<tr>
 +
 +
<td></td>
 +
<td>Amount Used</td>
 +
<td>Amount</td>
 +
<td>A 260/280</td>
 +
<td>A 260/230</td>
 +
 
 +
</tr>
 +
 
 +
</thead>
 +
 
 +
<tbody>
 +
<tr>
 +
<td>P1 (luxPR promoter) </br>(BBa_R6002) </td>
 +
<td>1 μl</td>
 +
<td>117.2ng/μl</td>
 +
<td>1.85</td>
 +
<td>1.54</td>
 +
</tr>
 +
</tbody>
 +
 
 +
  </table>
 +
 
 +
</div>
 +
</div>
 +
 
 +
<div class="notebook-data-box1">
 +
<h2> Week Three ( July 21 - July 26 )</h2>
 +
<div class="panel panel-success">
 +
  <div class="panel-heading">
 +
    <h3 class="panel-title" id="21-july">21st July 2015</h3>
 +
  </div>
 +
 
 +
  <ul class="list-group">
 +
    <li class="list-group-item">Cells containing LuxR were inoculated in 10 ml culture and kept overnight.</li>
 +
  </ul>
 +
</div>
 +
 
 +
<div class="panel panel-success">
 +
  <div class="panel-heading">
 +
    <h3 class="panel-title" id="22-july">22nd July 2015</h3>
 +
  </div>
 +
 
 +
 
 +
  <ul class="list-group">
 +
    <li class="list-group-item">LuxR DNA was extracted following the protocol of H1 Media.</li>
 +
    <li class="list-group-item">Gel electrophoresis done for LuxR DNA</li>
 +
    <li class="list-group-item">Nanodrop done for LuxR.</li>
 +
  </ul>
 +
 
 +
  <table class="table">
 +
    <thead>
 +
 +
<tr>
 +
 +
<td></td>
 +
<td>Amount Used</td>
 +
<td>Amount</td>
 +
<td>A 260/280</td>
 +
<td>A 260/230</td>
 +
 
 +
</tr>
 +
 
 +
</thead>
 +
 
 +
<tbody>
 +
<tr>
 +
<td>P3  (LuxR) </br> (BBa_K546003)</td>
 +
<td>1μl</td>
 +
<td>191.2ng/μl</td>
 +
<td>1.91</td>
 +
<td>2.14</td>
 +
</tr>
 +
</tbody>
 +
 
 +
  </table>
 +
 
 +
  <ul class="list-group">
 +
    <li class="list-group-item">Restriction digestion mix. used for LuxR DNA extracted before.  Restriction enzymes used: </br> 1. Xba I </br> 2. Pst I</li>
 +
  </ul>
 +
 
 +
  <table class="table">
 +
    <thead>
 +
 +
<tr>
 +
 +
<td class="well">Reaction Mixture</td>
 +
 
 +
</tr>
 +
 
 +
</thead>
 +
 
 +
<tbody>
 +
<tr>
 +
<td>DNA</td>
 +
<td>10μl</td>
 +
</tr>
 +
 
 +
<tr>
 +
<td>Buffer</td>
 +
<td>2μl (3:1)</td>
 +
</tr>
 +
 
 +
<tr>
 +
<td>Pst 1</td>
 +
<td>1μl</td>
 +
</tr>
 +
 
 +
<tr>
 +
<td>Xba 1</td>
 +
<td>1μl</td>
 +
</tr>
 +
 
 +
<tr>
 +
<td>H2O</td>
 +
<td>6μl</td>
 +
</tr>
 +
 
 +
<tr>
 +
 +
<td class="well">Kept at 37 degree Centigrade overnight.</td>
 +
 
 +
</tr>
 +
 
 +
</tbody>
 +
 
 +
  </table>
 +
 
 +
  <ul class="list-group">
 +
    <li class="list-group-item">To check the growth of crt EBI (reporter) in Ampicillin + LB , 3ml culture grown overnight.</li>
 +
  </ul>
 +
 
 +
</div>
 +
 
 +
<div class="panel panel-success">
 +
  <div class="panel-heading">
 +
    <h3 class="panel-title" id="23-july">23rd July 2015</h3>
 +
  </div>
 +
 
 +
 
 +
  <ul class="list-group">
 +
    <li class="list-group-item">Glycerol stocks made from 3 ml culture of crt EBI reporter which was grown overnight. (20% glycerol stock kept in 20℃)</li>
 +
    <li class="list-group-item">Gel run for restriction digestion of LuxR.</li>
 +
    <li class="list-group-item">Inoculation and streaking done for crt EBI reporter.</li>
 +
  </ul>
 +
</div>
 +
 
 +
<div class="panel panel-success">
 +
  <div class="panel-heading">
 +
    <h3 class="panel-title" id="24-july">24th July 2015</h3>
 +
  </div>
 +
 
 +
 
 +
  <ul class="list-group">
 +
    <li class="list-group-item">Plasmid preparation for crt easeInOutCubic</li>
 +
    <li class="list-group-item well">Precautions: </br>1. Use only one column for 10 ml culture while extracting.
 +
                    </br>2. After loading the supernatant in the column, wait for 10 min for better DNA binding.
 +
                    </br>3. Before eluting with (distilled) autoclaved water wait for 10 min. for better elution</li>
 +
    <li class="list-group-item">Nanodrop for crt EBI</li>
 +
  </ul>
 +
</div>
 +
 
 +
<table class="table">
 +
    <thead>
 +
 +
<tr>
 +
 +
<td></td>
 +
<td>Amount Used</td>
 +
<td>Amount</td>
 +
<td>A 260/280</td>
 +
<td>A 260/230</td>
 +
 
 +
</tr>
 +
 
 +
</thead>
 +
 
 +
<tbody>
 +
<tr>
 +
<td>P2 (crt EBI) </br>(BBa_K274100) </td>
 +
<td>1μl</td>
 +
<td>284.2ng/μ</td>
 +
<td>1.86</td>
 +
<td>2.14</td>
 +
</tr>
 +
</tbody>
 +
 
 +
  </table>
 +
 
 +
  <ul class="list-group">
 +
    <li class="list-group-item">Reaction mixture for restriction digestion of crt EBI</li>
 +
  </ul>
 +
 
 +
  <table class="table">
 +
 
 +
<tbody>
 +
<tr>
 +
<td>DNA</td>
 +
<td>4μl</td>
 +
</tr>
 +
 
 +
<tr>
 +
<td>Buffer</td>
 +
<td>2μl (3:1)</td>
 +
</tr>
 +
 
 +
<tr>
 +
<td>Pst 1</td>
 +
<td>1μl</td>
 +
</tr>
 +
 
 +
<tr>
 +
<td>Xba 1</td>
 +
<td>1μl</td>
 +
</tr>
 +
 
 +
<tr>
 +
<td>H2O</td>
 +
<td>12μl</td>
 +
</tr>
 +
 
 +
</tbody>
 +
 
 +
  </table>
 +
 
 +
<div class="panel panel-success">
 +
  <div class="panel-heading">
 +
    <h3 class="panel-title" id="26-july">26th July 2015</h3>
 +
  </div>
 +
 
 +
  <ul class="list-group">
 +
    <li class="list-group-item">50 ml culture used for extracting luxPR plasmid using E. coli DH5ɑ strain</li>
 +
    <li class="list-group-item">34 μl/ml of antibiotic (camR) used as a resistance marker</li>
 +
    <li class="list-group-item">Plasmid extracted for the LuxPR part from 50 ml culture. 60 μl volume of plasmid obtained.</li>
 +
  </ul>
 +
 
 +
  <table class="table">
 +
    <thead>
 +
 +
<tr>
 +
 +
<td></td>
 +
<td>Amount Used</td>
 +
<td>Amount</td>
 +
<td>A 260/280</td>
 +
<td>A 260/230</td>
 +
 
 +
</tr>
 +
 
 +
</thead>
 +
 
 +
<tbody>
 +
<tr>
 +
<td>P1  (LuxPR) </br>(BBa_R6002) </td>
 +
<td>1μl</td>
 +
<td>170 ng/μl</td>
 +
<td>1.83</td>
 +
<td>2.05</td>
 +
</tr>
 +
</tbody>
 +
 
 +
  </table>
 +
 
 +
  <ul class="list-group">
 +
    <li class="list-group-item well">Restriction digestion of LuxPR and crt EBI</li>
 +
  </ul>
 +
 
 +
  <table class="table">
 +
 
 +
<tbody>
 +
<tr>
 +
<td></td>
 +
<td>LuxPR</td>
 +
<td>crtEBI</td>
 +
</tr>
 +
 
 +
<tr>
 +
<td>DNA</td>
 +
<td>8μl</td>
 +
<td>6μl</td>
 +
</tr>
 +
 
 +
<tr>
 +
<td>Enzyme 1</td>
 +
<td>1 μl                (Spe I)</td>
 +
<td>1μl                        (Xba I)</td>
 +
</tr>
 +
 
 +
<tr>
 +
<td>Enzyme 2</td>
 +
<td>1 μl                (Pst I) </td>
 +
<td>1μl                        (Pst I)</td>
 +
</tr>
 +
 
 +
<tr>
 +
<td>Buffer</td>
 +
<td>2 μl                (2:1)</td>
 +
<td>2μl                        (3:1)</td>
 +
</tr>
 +
 
 +
<tr>
 +
<td>H2O</td>
 +
<td>8μl</td>
 +
<td>10μl</td>
 +
</tr>
 +
 
 +
</tbody>
 +
 
 +
  </table>
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
</div>
 +
</div>
 +
 
 +
<div class="notebook-data-box1">
 +
<h2> WEEK Four( July 28 - August 1 )</h2>
 +
<div class="panel panel-success">
 +
  <div class="panel-heading">
 +
    <h3 class="panel-title" id="28-july">28th July 2015</h3>
 +
  </div>
 +
 
 +
 
 +
  <ul class="list-group">
 +
    <li class="list-group-item">Gel was run with crtEBI containing digested plasmid and LuxPR containing digested plasmid </br> Length of crtEBI  ~  3.3 kbp </br>Length of LuxPR ~  50 bp </br> Plasmid containing LuxPR  ~ 2.2 kbp
 +
    </li>
 +
    <li class="list-group-item">crtEBI part , and LuxPR containing part extracted from gel . DNA eluted using the kit </li>
 +
    <li class="list-group-item">Nanodrop was run</li>
 +
  </ul>
 +
 
 +
  <table class="table">
 +
 
 +
<tbody>
 +
<tr>
 +
<td></td>
 +
<td>Amount used</td>
 +
<td>amount</td>
 +
</tr>
 +
 
 +
<tr>
 +
<td>LuxPR</td>
 +
<td>1μl</td>
 +
<td>28 ng/μl</td>
 +
</tr>
 +
 
 +
<tr>
 +
<td>crtEBI</td>
 +
<td>1μl</td>
 +
<td>50 ng/μl</td>
 +
</tr>
 +
 
 +
</tbody>
 +
 
 +
  </table>
 +
 
 +
  <ul class="list-group">
 +
    <li class="list-group-item">Ligation performed of crtEBI part into plasmid backbone containing LuxPR. </br> Insert:  crEBI (3.3 kb) , 50 ng/μl </br> Vector:  LuxPR (2.1 kb) , 28 ng/μl
 +
    </li>
 +
  </ul>
 +
 
 +
</div>
 +
 
 +
<table class="table">
 +
 
 +
<tbody>
 +
<tr>
 +
<td>Vector</td>
 +
<td>6 μl        (170 ng)</td>
 +
</tr>
 +
 
 +
<tr>
 +
<td>Insert</td>
 +
<td>2 μl          (89 ng)</td>
 +
</tr>
 +
 
 +
<tr>
 +
<td>T4 ligase buffer</td>
 +
<td>2 μl</td>
 +
</tr>
 +
 
 +
<tr>
 +
<td>T4 ligase</td>
 +
<td>1 μl</td>
 +
</tr>
 +
 
 +
<tr>
 +
<td>H2O</td>
 +
<td>9 μl</td>
 +
</tr>
 +
 
 +
</tbody>
 +
 
 +
  </table>
 +
 
 +
<div class="panel panel-success">
 +
  <div class="panel-heading">
 +
    <h3 class="panel-title" id="29-july">29th July 2015</h3>
 +
  </div>
 +
 
 +
 
 +
  <ul class="list-group">
 +
    <li class="list-group-item">Transformed the ligation product of LuxPR and crtEBI(reporter gene). transformation procedure</li>
 +
    <li class="list-group-item">Plating done for the above construct.</li>
 +
  </ul>
 +
</div>
 +
 
 +
<div class="panel panel-success">
 +
  <div class="panel-heading">
 +
    <h3 class="panel-title" id="30-july">30th July 2015</h3>
 +
  </div>
 +
 
 +
 
 +
  <ul class="list-group">
 +
    <li class="list-group-item">No growth observed for the construct. Steps repeated.</li>
 +
    <li class="list-group-item">Restriction digestion of LuxPR: (single digestion)</li>
 +
   
 +
  </ul>
 +
 
 +
  <table class="table">
 +
 
 +
<tbody>
 +
<tr>
 +
<td>LuxPR</td>
 +
<td>7 μl</td>
 +
</tr>
 +
 
 +
<tr>
 +
<td>Spe I</td>
 +
<td>1 μl</td>
 +
</tr>
 +
 
 +
<tr>
 +
<td>Buffer (cutsmart)</td>
 +
<td>2 μl</td>
 +
</tr>
 +
 
 +
<tr>
 +
<td>H2O</td>
 +
<td>10 μl</td>
 +
</tr>
 +
 
 +
<tr>
 +
<td>Kept at 37℃ for 5-6 hours.</td>
 +
</tr>
 +
 
 +
</tbody>
 +
 
 +
  </table>
 +
 
 +
  <ul class="list-group">
 +
    <li class="list-group-item">Second digestion setup made.</li>
 +
  </ul>
 +
 
 +
  <table class="table">
 +
 
 +
  <tbody>
 +
<tr>
 +
<td>Product from previous digestion</td>
 +
<td>20 μl</td>
 +
</tr>
 +
 
 +
<tr>
 +
<td>Pst I</td>
 +
<td>1 μl</td>
 +
</tr>
 +
 
 +
<tr>
 +
<td>Buffer (3:1)</td>
 +
<td>2 μl</td>
 +
</tr>
 +
 
 +
<tr>
 +
<td>Kept at 37℃ for 5-6 hours.</td>
 +
</tr>
 +
 
 +
</tbody>
 +
 
 +
</table>
 +
 
 +
</div>
 +
 
 +
  <div class="panel panel-success">
 +
  <div class="panel-heading">
 +
    <h3 class="panel-title" id="31-july">31st July 2015</h3>
 +
  </div>
 +
 
 +
  <ul class="list-group">
 +
    <li class="list-group-item">Gel was run for the restriction digest product.</li>
 +
    <li class="list-group-item">Band was cut and DNA eluted using gel extraction kit.</li>
 +
    <li class="list-group-item">Ligation set up after purifying the DNA.</li>
 +
  </ul>
 +
</div>
 +
 
 +
<div class="panel panel-success">
 +
 +
  <div class="panel-heading">
 +
    <h3 class="panel-title" id="1-august">1st August 2015</h3>
 +
  </div>
 +
 
 +
  <ul class="list-group">
 +
    <li class="list-group-item">Transformation done for ligated product. Transformation procedure</li>
 +
  </ul>
 +
 
 +
</div>
 +
</div>
 +
 
 +
 
 +
</div>
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IIT Kharagpur</a>
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Revision as of 09:01, 18 September 2015

July

Mo Tu We Th Fr Sa Su
29 30 1 2 3 4 5
<div>6</div> <div>7</div> <div>8</div> <div>9</div> <div>10</div> 11 12
13 14 15 16 17 18 19
20 <div>21</div> <div>22</div> <div>23</div> <div>24</div> 25
26
27 <div>28</div> <div>29</div> <div>30</div> <div>31</div> 1 2
3 4 5 6 7 8 9

August

Mo Tu We Th Fr Sa Su
27 28 29 30 31 <div>1</div> 2
3 4 5 6 7 8 9
10 11 12 13 14 15 16
17 18 19 20 21 22 23
24 25 26 27 28 29 30
31 1 2 3 4 5 6

September

Mo Tu We Th Fr Sa Su
31 1 2 3 4 5 6
7 8 9 10 11 12 13
14 15 16 17 18 19 20
21 22 23 24 25 26 27
28 29 30 1 2 3 4
5 6 7 8 9 10 11

Week One ( July 6 - July 10 )

6th July 2015

  • LB media prepared (500 ml), autoclaved and stored at 4 degrees.
  • 250 ml of LB agar prepared and poured into the plates (chloramphenicol antibiotic used- concentration 1 micro litre/ml)

7th July 2015

  • DNA from wells dissolved into 10 μl distilled water. DNA kit plate instructions
  • Transformed 4μl of DNA into 100μl of E.Coli DH5ɑ. transformation procedure)
  • 100μl of transformed cells were plated on chloramphenicol resistant plates.and left overnight to grow.

8th July 2015

  • Colonies for luxPR promoter (BBa_R6002) constructs were observed. A colony was picked and inoculated in 3 ml culture for glycerol stock preparation.
  • No colony observed for luxR protein (BBa_K546003) construct.

9th July 2015

  • Transformation repeated for luxR protein construct .transformation procedure
  • Culture prepared for plasmid isolation for luxPR promoter. Colony was picked and inoculated in 10 ml culture.

10th July 2015

  • No colony observed for luxR protein construct
  • Plasmid isolation done for luxPR promoter
  • Gel electrophoresis done for luxPR promoter
  • Lane 7 : 1kb marker
    Lane 8 : luxPR promoter
  • Gel electrophoresis done for luxPR promoter
Amount Used Amount A 260/280 A 260/230
P1 (luxPR promoter)
(BBa_R6002) 		1 μl 117.2ng/μl 1.85 1.54

Week Three ( July 21 - July 26 )

21st July 2015

  • Cells containing LuxR were inoculated in 10 ml culture and kept overnight.

22nd July 2015

  • LuxR DNA was extracted following the protocol of H1 Media.
  • Gel electrophoresis done for LuxR DNA
  • Nanodrop done for LuxR.
Amount Used Amount A 260/280 A 260/230
P3 (LuxR)
(BBa_K546003)		 1μl 191.2ng/μl 1.91 2.14
  • Restriction digestion mix. used for LuxR DNA extracted before. Restriction enzymes used:
    1. Xba I
    2. Pst I
Reaction Mixture
DNA 10μl
Buffer 2μl (3:1)
Pst 1 1μl
Xba 1 1μl
H2O 6μl
Kept at 37 degree Centigrade overnight.
  • To check the growth of crt EBI (reporter) in Ampicillin + LB , 3ml culture grown overnight.

23rd July 2015

  • Glycerol stocks made from 3 ml culture of crt EBI reporter which was grown overnight. (20% glycerol stock kept in 20℃)
  • Gel run for restriction digestion of LuxR.
  • Inoculation and streaking done for crt EBI reporter.

24th July 2015

  • Plasmid preparation for crt easeInOutCubic
  • Precautions:
    1. Use only one column for 10 ml culture while extracting.
    2. After loading the supernatant in the column, wait for 10 min for better DNA binding.
    3. Before eluting with (distilled) autoclaved water wait for 10 min. for better elution
  • Nanodrop for crt EBI
Amount Used Amount A 260/280 A 260/230
P2 (crt EBI)
(BBa_K274100) 		1μl 284.2ng/μ 1.86 2.14
  • Reaction mixture for restriction digestion of crt EBI
DNA 4μl
Buffer 2μl (3:1)
Pst 1 1μl
Xba 1 1μl
H2O 12μl

26th July 2015

  • 50 ml culture used for extracting luxPR plasmid using E. coli DH5ɑ strain
  • 34 μl/ml of antibiotic (camR) used as a resistance marker
  • Plasmid extracted for the LuxPR part from 50 ml culture. 60 μl volume of plasmid obtained.
Amount Used Amount A 260/280 A 260/230
P1 (LuxPR)
(BBa_R6002) 		1μl 170 ng/μl 1.83 2.05
  • Restriction digestion of LuxPR and crt EBI
LuxPR crtEBI
DNA 8μl 6μl
Enzyme 1 1 μl (Spe I) 1μl (Xba I)
Enzyme 2 1 μl (Pst I) 1μl (Pst I)
Buffer 2 μl (2:1) 2μl (3:1)
H2O 8μl 10μl

WEEK Four( July 28 - August 1 )

28th July 2015

  • Gel was run with crtEBI containing digested plasmid and LuxPR containing digested plasmid
    Length of crtEBI ~ 3.3 kbp
    Length of LuxPR ~ 50 bp
    Plasmid containing LuxPR ~ 2.2 kbp
  • crtEBI part , and LuxPR containing part extracted from gel . DNA eluted using the kit
  • Nanodrop was run
Amount used amount
LuxPR 1μl 28 ng/μl
crtEBI 1μl 50 ng/μl
  • Ligation performed of crtEBI part into plasmid backbone containing LuxPR.
    Insert: crEBI (3.3 kb) , 50 ng/μl
    Vector: LuxPR (2.1 kb) , 28 ng/μl
Vector 6 μl (170 ng)
Insert 2 μl (89 ng)
T4 ligase buffer 2 μl
T4 ligase 1 μl
H2O 9 μl

29th July 2015

  • Transformed the ligation product of LuxPR and crtEBI(reporter gene). transformation procedure
  • Plating done for the above construct.

30th July 2015

  • No growth observed for the construct. Steps repeated.
  • Restriction digestion of LuxPR: (single digestion)
LuxPR 7 μl
Spe I 1 μl
Buffer (cutsmart) 2 μl
H2O 10 μl
Kept at 37℃ for 5-6 hours.
  • Second digestion setup made.
Product from previous digestion 20 μl
Pst I 1 μl
Buffer (3:1) 2 μl
Kept at 37℃ for 5-6 hours.

31st July 2015

  • Gel was run for the restriction digest product.
  • Band was cut and DNA eluted using gel extraction kit.
  • Ligation set up after purifying the DNA.

1st August 2015

  • Transformation done for ligated product. Transformation procedure
Department of Biotechnology
IIT Kharagpur

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