Difference between revisions of "Team:IIT Kharagpur/Notebook"
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<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/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 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> | ||
− | + | <ul> | |
+ | <li><a href="https://2015.igem.org/Team:IIT_Kharagpur/Practices#meetups">Meetups</li> | ||
+ | <li><a href="https://2015.igem.org/Team:IIT_Kharagpur/Practices#collabs">Collaborations</li> | ||
+ | <li><a href="https://2015.igem.org/Team:IIT_Kharagpur/Practices#surveys">Surveys</a></li> | ||
+ | <li><a href="https://2015.igem.org/Team:IIT_Kharagpur/Practices#interviews">Interviews</a></li> | ||
+ | <li><a href="https://2015.igem.org/Team:IIT_Kharagpur/Practices#workshops">Workshops</a></li> | ||
+ | <li><a href="https://2015.igem.org/Team:IIT_Kharagpur/Practices#guidebook">Guidebook</a></li> | ||
+ | </ul> | ||
</li> | </li> | ||
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<div class="notebook-text-div1"> | <div class="notebook-text-div1"> | ||
− | <img class="notebook-text1"src=" | + | <img class="notebook-text1"src="https://static.igem.org/mediawiki/2015/b/bc/IIT_Kharagpur_notebook-text.png" /> |
</div> | </div> | ||
<div class="notebook-text-div2"> | <div class="notebook-text-div2"> | ||
− | <a href="#"><img class="notebook-text2"src=" | + | <a href="#"><img class="notebook-text2"src="https://static.igem.org/mediawiki/2015/1/18/IIT_Kharagpur_notebook-icon.png" /></a> |
</div> | </div> | ||
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<div class="orange"></div> | <div class="orange"></div> | ||
</div> | </div> | ||
+ | <div class="body-wrapper" style="background:url('https://static.igem.org/mediawiki/2015/7/70/IIT_Kharagpur_bg_pattern_swirl.png')"> | ||
<div class="container notebook-mid"> | <div class="container notebook-mid"> | ||
<div class="row"> | <div class="row"> | ||
Line 483: | Line 491: | ||
</ul> | </ul> | ||
− | + | ||
− | + | ||
− | + | ||
<ul class="list-group"> | <ul class="list-group"> | ||
Line 547: | Line 553: | ||
<table class="table"> | <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> | ||
+ | |||
+ | |||
+ | <table class="table"> | ||
<thead> | <thead> | ||
Line 1,004: | Line 1,038: | ||
</div> | </div> | ||
+ | </div> | ||
+ | |||
+ | <div class="notebook-data-box1"> | ||
+ | <h2> Week Five ( August 2 - August 7)</h2> | ||
+ | <div class="panel panel-success"> | ||
+ | <div class="panel-heading"> | ||
+ | <h3 class="panel-title" id="2-august">2 August</h3> | ||
+ | </div> | ||
+ | |||
+ | |||
+ | <ul class="list-group"> | ||
+ | <li class="list-group-item">A single colony was observed.</li> | ||
+ | <li class="list-group-item">The colony was inoculated into a 3 ml Chloramphenicol containing medium.</li> | ||
+ | </ul> | ||
+ | </div> | ||
+ | |||
+ | <div class="panel panel-success"> | ||
+ | <div class="panel-heading"> | ||
+ | <h3 class="panel-title" id="3-august">3rd August</h3> | ||
+ | </div> | ||
+ | |||
+ | |||
+ | <ul class="list-group"> | ||
+ | <li class="list-group-item">Plasmid extraction done for the colony.</li> | ||
+ | <li class="list-group-item">Screening done by running Gel. The band obtained was below 2 kb size.( Expected band size was 5.4 kb)</li> | ||
+ | |||
+ | </ul> | ||
+ | </div> | ||
+ | |||
+ | <div class="panel panel-success"> | ||
+ | <div class="panel-heading"> | ||
+ | <h3 class="panel-title" id="6-august">6th August</h3> | ||
+ | </div> | ||
+ | |||
+ | |||
+ | <ul class="list-group"> | ||
+ | <li class="list-group-item">Double digestion performed with Spe I and Pst I.</li> | ||
+ | |||
+ | </ul> | ||
+ | <table class="table" id="week-four"> | ||
+ | <caption>Reaction Mixture</caption> | ||
+ | |||
+ | |||
+ | <tbody> | ||
+ | <tr> | ||
+ | <td>DNA + Water 20 μl</td> | ||
+ | <td>20 μl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Spe I</td> | ||
+ | <td>0.5 μl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Pst I</td> | ||
+ | <td>1.0 μl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Buffer (2:1)</td> | ||
+ | <td>2.0 μl</td> | ||
+ | </tr> | ||
+ | </tbody> | ||
+ | |||
+ | </table> | ||
+ | </div> | ||
+ | |||
+ | <div class="panel panel-success"> | ||
+ | <div class="panel-heading"> | ||
+ | <h3 class="panel-title" id="7-august">7th August</h3> | ||
+ | </div> | ||
+ | |||
+ | |||
+ | <ul class="list-group"> | ||
+ | <li class="list-group-item">Digested Lux PR eluted from gel.</li> | ||
+ | <li class="list-group-item">Plasmid extraction done for Lux PR. Concentration: 126.5 ng/ μl </br>Volume: 55 μl</li> | ||
+ | <li class="list-group-item">Ligation reaction mixture for Lux PR (27 ng/μl) + crtEBI (28 ng/μl)</li> | ||
+ | </ul> | ||
+ | |||
+ | <table class="table" id="week-four"> | ||
+ | <caption>Reaction Mixture</caption> | ||
+ | |||
+ | |||
+ | <tbody> | ||
+ | <tr> | ||
+ | <td></td> | ||
+ | <td>V:I = 3:1</td> | ||
+ | <td>V:I=2:1</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>crtBI (28.2 ng/μl)</td> | ||
+ | <td>3μl</td> | ||
+ | <td>4μl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>luxPR (27.2 ng/μl)</td> | ||
+ | <td>6μl</td> | ||
+ | <td>6μl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>T DNA ligase</td> | ||
+ | <td>1.5μl</td> | ||
+ | <td>1.μl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>ligase buffer</td> | ||
+ | <td>1.5μl</td> | ||
+ | <td>1.5μl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Water</td> | ||
+ | <td>8μl</td> | ||
+ | <td>7μl</td> | ||
+ | </tr> | ||
+ | </tbody> | ||
+ | |||
+ | </table> | ||
+ | <caption>ligation @ 16 for 16 hours.</caption> | ||
+ | <table class="table" id="week-four"> | ||
+ | |||
+ | |||
+ | |||
+ | <tbody> | ||
+ | <tr> | ||
+ | <td>crtEBI</td> | ||
+ | <td>3μl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>luxPR</td> | ||
+ | <td>6μl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>buffer</td> | ||
+ | <td>1.5μl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>ligase</td> | ||
+ | <td>1.2μl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>water</td> | ||
+ | <td>3.3μl</td> | ||
+ | </tr> | ||
+ | </tbody> | ||
+ | |||
+ | </table> | ||
+ | <caption>Digestion: | ||
+ | |||
+ | LuxPR (E and S) 3μl CrtEBI (E and X) 1.4 μl</caption> | ||
+ | <table class="table" id="week-four"> | ||
+ | |||
+ | |||
+ | |||
+ | <tbody> | ||
+ | <tr> | ||
+ | <td>DNA</td> | ||
+ | <td>12μl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>EcoRI</td> | ||
+ | <td>1μl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Buffer</td> | ||
+ | <td>2μl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>H2O</td> | ||
+ | <td>5μl</td> | ||
+ | </tr> | ||
+ | |||
+ | </tbody> | ||
+ | |||
+ | </table> | ||
+ | <caption>Sequential</caption> | ||
+ | |||
+ | |||
+ | <table class="table" id="week-four"> | ||
+ | |||
+ | |||
+ | |||
+ | <tbody> | ||
+ | <tr> | ||
+ | <td>DNA</td> | ||
+ | <td>10μl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>EcoRI</td> | ||
+ | <td>1μl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Buffer [Neb buffer EcoRI]</td> | ||
+ | <td>2μl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>H2O</td> | ||
+ | <td>7μl</td> | ||
+ | </tr> | ||
+ | |||
+ | </tbody> | ||
+ | |||
+ | </table> | ||
+ | |||
+ | <caption>Digestion 2</caption> | ||
+ | |||
+ | |||
+ | <table class="table" id="week-four"> | ||
+ | |||
+ | |||
+ | |||
+ | <tbody> | ||
+ | <tr> | ||
+ | <td>LuxPR</td> | ||
+ | <td>17μl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Spe I</td> | ||
+ | <td>1μl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Buffer (Cutsmart)</td> | ||
+ | <td>2μl</td> | ||
+ | </tr> | ||
+ | |||
+ | |||
+ | </tbody> | ||
+ | |||
+ | </table> | ||
+ | |||
+ | <table class="table" id="week-four"> | ||
+ | |||
+ | |||
+ | |||
+ | <tbody> | ||
+ | <tr> | ||
+ | <td>CrtEBI</td> | ||
+ | <td>17μl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Xba I</td> | ||
+ | <td>1μl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Buffer (Cutsmart)</td> | ||
+ | <td>2μl</td> | ||
+ | </tr> | ||
+ | |||
+ | |||
+ | </tbody> | ||
+ | |||
+ | </table> | ||
+ | |||
+ | <caption>Result:</br> | ||
+ | |||
+ | 2% gel was prepared since the LuxPR insert was only 55 bp. The gel wasn’t prepared properly. | ||
+ | |||
+ | The same bands were observed twice. This was probably because the concentration was not | ||
+ | |||
+ | uniform throughout the height of the gel. The bands ran faster at the upper surface and slower | ||
+ | |||
+ | at the lower surface. | ||
+ | |||
+ | The 55 bp was not observed at all. | ||
+ | </caption> | ||
+ | <caption>3 A Assembly</br> | ||
+ | |||
+ | Restriction Digestion:</br> | ||
+ | |||
+ | FIRST DIGESTION</br> | ||
+ | |||
+ | Part A</caption> | ||
+ | <table class="table" id="week-four"> | ||
+ | |||
+ | |||
+ | |||
+ | <tbody> | ||
+ | <tr> | ||
+ | <td>LuxPR (128 ng/μl)</td> | ||
+ | <td>3μl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Buffer (EcoRI Buffer)</td> | ||
+ | <td>2μl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>EcoRI</td> | ||
+ | <td>1μl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>N.F./ dd H2O</td> | ||
+ | <td>14μl</td> | ||
+ | </tr> | ||
+ | |||
+ | </tbody> | ||
+ | |||
+ | </table> | ||
+ | |||
+ | <table class="table" id="week-four"> | ||
+ | <caption>Part B</caption> | ||
+ | |||
+ | |||
+ | <tbody> | ||
+ | <tr> | ||
+ | <td>CrtEBI (140 ng/μl )</td> | ||
+ | <td>3μl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Buffer (2:1)</td> | ||
+ | <td>2μl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Xba I</td> | ||
+ | <td>1μl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>N.F. H2O</td> | ||
+ | <td>14μl</td> | ||
+ | </tr> | ||
+ | |||
+ | </tbody> | ||
+ | |||
+ | </table> | ||
+ | <caption>A and B kept at 37 for 2 hours</br> | ||
+ | |||
+ | Then PCR Purified.</br> | ||
+ | |||
+ | Backbone</caption> | ||
+ | |||
+ | <table class="table" id="week-four"> | ||
+ | |||
+ | |||
+ | |||
+ | <tbody> | ||
+ | <tr> | ||
+ | <td>DNA</td> | ||
+ | <td>5μl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>EcoRI</td> | ||
+ | <td>0.5μl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Pst I</td> | ||
+ | <td>0.5μl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Dpa I</td> | ||
+ | <td>0.5μl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Buffer (2:1)</td> | ||
+ | <td>2μl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>N.F. H2O</td> | ||
+ | <td>11.5μl</td> | ||
+ | </tr> | ||
+ | |||
+ | </tbody> | ||
+ | |||
+ | </table> | ||
+ | <caption> | ||
+ | |||
+ | Kept at 37 for 1 hour</br> | ||
+ | |||
+ | PCR Purification</br> | ||
+ | |||
+ | SECOND DIGESTION</br> | ||
+ | </caption> | ||
+ | |||
+ | <table class="table" id="week-four"> | ||
+ | |||
+ | |||
+ | |||
+ | <tbody> | ||
+ | <tr> | ||
+ | <td>DNA (LuxPR)</td> | ||
+ | <td>20μl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Spe I</td> | ||
+ | <td>1μl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Buffer (Cutsmart)</td> | ||
+ | <td>2μl</td> | ||
+ | </tr> | ||
+ | |||
+ | |||
+ | </tbody> | ||
+ | |||
+ | </table> | ||
+ | |||
+ | <table class="table" id="week-four"> | ||
+ | |||
+ | |||
+ | |||
+ | <tbody> | ||
+ | <tr> | ||
+ | <td>DNA (CrtEBI)</td> | ||
+ | <td>20μl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Pst I</td> | ||
+ | <td>1μl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Buffer (3:1)</td> | ||
+ | <td>2μl</td> | ||
+ | </tr> | ||
+ | |||
+ | |||
+ | </tbody> | ||
+ | |||
+ | </table> | ||
+ | <caption> | ||
+ | Nanodrop data:</br> | ||
+ | |||
+ | LuxPR 15 ng/μl </br> | ||
+ | |||
+ | CrtEBI 15 ng/μl </br> | ||
+ | |||
+ | Backbone 5 ng/μl </br> | ||
+ | |||
+ | Size of plasmids:</br> | ||
+ | |||
+ | LuxPR 2.1 </br> | ||
+ | |||
+ | CrtEBI 5.5 </br> | ||
+ | |||
+ | Backbone 2.2</br> | ||
+ | |||
+ | Ligation Mixture:</br> | ||
+ | |||
+ | Molar Ratio LuxPR : CrtEBI : Backbone = 1 : 1 : 0.5 (Recommended)</br> | ||
+ | </caption> | ||
+ | <table class="table" id="week-four"> | ||
+ | |||
+ | |||
+ | |||
+ | <tbody> | ||
+ | <tr> | ||
+ | <td>LuxPR</td> | ||
+ | <td>3μl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>CrtEBI</td> | ||
+ | <td>8μl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Backbone</td> | ||
+ | <td>5μl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Buffer</td> | ||
+ | <td>2μl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Ligase (T4)</td> | ||
+ | <td>1μl</td> | ||
+ | </tr> | ||
+ | |||
+ | |||
+ | </tbody> | ||
+ | |||
+ | </table> | ||
+ | <caption> | ||
+ | |||
+ | Molar Ratio LuxPR : CrtEBI : Backbone = 3 : 1 : 0.5 | ||
+ | </caption> | ||
+ | |||
+ | <table class="table" id="week-four"> | ||
+ | |||
+ | |||
+ | |||
+ | <tbody> | ||
+ | <tr> | ||
+ | <td>LuxPR</td> | ||
+ | <td>6μl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>CrtEBI</td> | ||
+ | <td>5μl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Backbone</td> | ||
+ | <td>3μl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Buffer</td> | ||
+ | <td>2μl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Ligase (T4)</td> | ||
+ | <td>1μl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Water (T4)</td> | ||
+ | <td>3μl</td> | ||
+ | </tr> | ||
+ | |||
+ | |||
+ | </tbody> | ||
+ | |||
+ | </table> | ||
+ | <caption>3A assembly possible plasmids and their sizes for screening | ||
+ | |||
+ | correct plasmid</caption> | ||
+ | </div> | ||
+ | |||
+ | <div class="panel panel-success"> | ||
+ | <div class="panel-heading"> | ||
+ | <h3 class="panel-title" id="17-august">17th August</h3> | ||
+ | </div> | ||
+ | |||
+ | <caption>1-10 b → 1:1:0.5 | ||
+ | |||
+ | 1-10 a→ 3:1:0.5 | ||
+ | |||
+ | 7b colony→ plasmid check (68 ng/ μl) | ||
+ | |||
+ | CLONE CONFIRMATION</br> | ||
+ | |||
+ | Digestion 1</caption> | ||
+ | |||
+ | <table class="table" id="week-four"> | ||
+ | |||
+ | |||
+ | |||
+ | <tbody> | ||
+ | <tr> | ||
+ | <td>DNA (68 ng)</td> | ||
+ | <td>7μl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Buffer (2:1)</td> | ||
+ | <td>2μl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>EcoRI</td> | ||
+ | <td>0.5μl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Pst I</td> | ||
+ | <td>0.5μl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>H2O</td> | ||
+ | <td>10μl</td> | ||
+ | </tr> | ||
+ | |||
+ | |||
+ | </tbody> | ||
+ | |||
+ | </table> | ||
+ | <caption>Digestion 2</caption> | ||
+ | <table class="table" id="week-four"> | ||
+ | |||
+ | |||
+ | |||
+ | <tbody> | ||
+ | <tr> | ||
+ | <td>DNA </td> | ||
+ | <td>7μl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Buffer (CS)</td> | ||
+ | <td>2μl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Xba I</td> | ||
+ | <td>0.5μl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Spe I</td> | ||
+ | <td>0.5μl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>H2O</td> | ||
+ | <td>10μl</td> | ||
+ | </tr> | ||
+ | |||
+ | |||
+ | </tbody> | ||
+ | |||
+ | </table> | ||
+ | |||
+ | <table class="table" id="week-four"> | ||
+ | |||
+ | |||
+ | |||
+ | <tbody> | ||
+ | <tr> | ||
+ | <td>DNA </td> | ||
+ | <td>7μl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Buffer (3:1)</td> | ||
+ | <td>2μl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Pst I</td> | ||
+ | <td>1μl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>H2O</td> | ||
+ | <td>10μl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Total</td> | ||
+ | <td>20μl</td> | ||
+ | </tr> | ||
+ | |||
+ | |||
+ | </tbody> | ||
+ | |||
+ | </table> | ||
+ | |||
+ | |||
+ | |||
+ | </div> | ||
+ | |||
+ | <div class="panel panel-success"> | ||
+ | <div class="panel-heading"> | ||
+ | <h3 class="panel-title" id="3-september">3rd September</h3> | ||
+ | </div> | ||
+ | |||
+ | <caption>3A Assembly (Attempt 4)</caption> | ||
+ | |||
+ | <table class="table" id="week-four"> | ||
+ | |||
+ | |||
+ | |||
+ | <tbody> | ||
+ | <tr> | ||
+ | <td>LuxR</td> | ||
+ | <td>7μl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>EcoRI</td> | ||
+ | <td>1μl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Buffer (EcoRI)</td> | ||
+ | <td>2μl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>dd H2O</td> | ||
+ | <td>10μl</td> | ||
+ | </tr> | ||
+ | |||
+ | |||
+ | |||
+ | </tbody> | ||
+ | |||
+ | </table> | ||
+ | |||
+ | <table class="table" id="week-four"> | ||
+ | |||
+ | |||
+ | |||
+ | <tbody> | ||
+ | <tr> | ||
+ | <td>CrtEBI</td> | ||
+ | <td>10μl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Xba I</td> | ||
+ | <td>1μl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Pst I</td> | ||
+ | <td>2μl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>dBuffer (3:1)</td> | ||
+ | <td>10μl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>dd H2O</td> | ||
+ | <td>10μl</td> | ||
+ | </tr> | ||
+ | |||
+ | |||
+ | |||
+ | </tbody> | ||
+ | |||
+ | </table> | ||
+ | <caption>Digestion 2</caption> | ||
+ | <table class="table" id="week-four"> | ||
+ | |||
+ | |||
+ | |||
+ | <tbody> | ||
+ | <tr> | ||
+ | <td>DNA </td> | ||
+ | <td>7μl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Buffer (CS)</td> | ||
+ | <td>2μl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Xba I</td> | ||
+ | <td>0.5μl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Spe I</td> | ||
+ | <td>0.5μl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>H2O</td> | ||
+ | <td>10μl</td> | ||
+ | </tr> | ||
+ | |||
+ | |||
+ | </tbody> | ||
+ | |||
+ | </table> | ||
+ | |||
+ | <table class="table" id="week-four"> | ||
+ | |||
+ | |||
+ | |||
+ | <tbody> | ||
+ | <tr> | ||
+ | <td>DNA </td> | ||
+ | <td>7μl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Buffer (3:1)</td> | ||
+ | <td>2μl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Pst I</td> | ||
+ | <td>1μl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>H2O</td> | ||
+ | <td>10μl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Total</td> | ||
+ | <td>20μl</td> | ||
+ | </tr> | ||
+ | |||
+ | |||
+ | </tbody> | ||
+ | |||
+ | </table> | ||
+ | |||
+ | |||
+ | |||
+ | </div> | ||
+ | <div class="panel panel-success"> | ||
+ | <div class="panel-heading"> | ||
+ | <h3 class="panel-title" id="8-september">8th September</h3> | ||
+ | </div> | ||
+ | |||
+ | <table class="table"> | ||
+ | |||
+ | <tbody> | ||
+ | <tr> | ||
+ | <td>DNA</td> | ||
+ | <td>13ul</td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td>Spe 1</td> | ||
+ | <td>1µl </td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td>Pst 1</td> | ||
+ | <td>1µl</td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td>Buffer</td> | ||
+ | <td>2ul</td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td>dd H2O</td> | ||
+ | <td>3ul</td> | ||
+ | </tr> | ||
+ | |||
+ | </tbody> | ||
+ | |||
+ | </table> | ||
+ | |||
+ | </div> | ||
+ | |||
+ | <div class="panel panel-success"> | ||
+ | <div class="panel-heading"> | ||
+ | <h3 class="panel-title" id="9-september">9th September</h3> | ||
+ | </div> | ||
+ | |||
+ | <table class="table"> | ||
+ | |||
+ | <tbody> | ||
+ | <tr> | ||
+ | <td>DNA(7b)</td> | ||
+ | <td>8µl</td> | ||
+ | </tr> | ||
+ | |||
+ | k <tr> | ||
+ | <td>Buffer (EcoR1)</td> | ||
+ | <td>2µl </td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td>dd H20</td> | ||
+ | <td>9µl</td> | ||
+ | </tbody> | ||
+ | |||
+ | </table> | ||
+ | <table class="table"> | ||
+ | |||
+ | <tbody> | ||
+ | <tr> | ||
+ | <td>DNA (CrtEBI)</td> | ||
+ | <td>10µl</td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td>Buffer (EcorR1)</td> | ||
+ | <td>2µl </td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td>dd H2O</td> | ||
+ | <td>7µl</td> | ||
+ | </tr> | ||
+ | |||
+ | </tbody> | ||
+ | |||
+ | </table> | ||
+ | |||
+ | <caption>(To get the backbone)</caption> | ||
+ | <table class="table"> | ||
+ | |||
+ | <tbody> | ||
+ | <tr> | ||
+ | <td>DNA (LuxR)</td> | ||
+ | <td>10µl</td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td>Buffer (EcorR1)</td> | ||
+ | <td>2µl </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>EcorR1</td> | ||
+ | <td>1µl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>dd H2O</td> | ||
+ | <td>7µl</td> | ||
+ | </tr> | ||
+ | |||
+ | </tbody> | ||
+ | |||
+ | </table> | ||
+ | <caption> | ||
+ | #Pst digestion : | ||
+ | #1μl Pst 2μl something | ||
+ | #Kept at 37℃ for 6 hours. | ||
+ | </caption> | ||
+ | </div> | ||
+ | |||
+ | <div class="panel panel-success"> | ||
+ | <div class="panel-heading"> | ||
+ | <h3 class="panel-title" id="10-september">10th September</h3> | ||
+ | </div> | ||
+ | <caption> | ||
+ | 7b : 12 ng/μl | ||
+ | LuxR : 12 ng/μl | ||
+ | LuxPR : 20 ng/μl | ||
+ | CrtEBI : 20 ng/μl | ||
+ | </caption> | ||
+ | |||
+ | <caption> Ligation 1 (standard assembly)</caption> | ||
+ | <table class="table"> | ||
+ | |||
+ | <tbody> | ||
+ | <tr> | ||
+ | <td>(X/P) CrtEBI (3.3)</td> | ||
+ | <td>7µl</td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td>(S/P) LuxPR (2.1)</td> | ||
+ | <td>5µl </td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td>T4 Ligase</td> | ||
+ | <td>1µl</td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td>Ligase Buffer</td> | ||
+ | <td>2µl</td> | ||
+ | </tr> </tbody> | ||
+ | |||
+ | </table> | ||
+ | <caption>Ligation 2 (breakthrough standard assembly)</caption> | ||
+ | <table class="table"> | ||
+ | |||
+ | <tbody> | ||
+ | <tr> | ||
+ | <td>(X/P) CrtEBI (3.3)</td> | ||
+ | <td>5µl</td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td>(S/P) Lux R (3.3)</td> | ||
+ | <td>8µl </td> | ||
+ | </tr> | ||
+ | |||
+ | <tr> | ||
+ | <td>T4 Ligase</td> | ||
+ | <td>1µl</td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td>Buffer</td> | ||
+ | <td>2µl</td> | ||
+ | </tr> | ||
+ | </tbody> | ||
+ | |||
+ | </table> | ||
+ | |||
+ | |||
+ | |||
+ | </div> | ||
</div> | </div> | ||
Line 1,012: | Line 1,980: | ||
</div> | </div> | ||
− | + | </div> | |
<div class="container-fluid footer1"> | <div class="container-fluid footer1"> |
Latest revision as of 00:23, 21 November 2015
July
Mo | Tu | We | Th | Fr | Sa | Su |
29 | 30 | 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 | 31 | 1 | 2 |
3 | 4 | 5 | 6 | 7 | 8 | 9 |
August
Mo | Tu | We | Th | Fr | Sa | Su |
27 | 28 | 29 | 30 | 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 |
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 |
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
Week Five ( August 2 - August 7)
2 August
- A single colony was observed.
- The colony was inoculated into a 3 ml Chloramphenicol containing medium.
3rd August
- Plasmid extraction done for the colony.
- Screening done by running Gel. The band obtained was below 2 kb size.( Expected band size was 5.4 kb)
6th August
- Double digestion performed with Spe I and Pst I.
DNA + Water 20 μl | 20 μl |
Spe I | 0.5 μl |
Pst I | 1.0 μl |
Buffer (2:1) | 2.0 μl |
7th August
- Digested Lux PR eluted from gel.
- Plasmid extraction done for Lux PR. Concentration: 126.5 ng/ μl Volume: 55 μl
- Ligation reaction mixture for Lux PR (27 ng/μl) + crtEBI (28 ng/μl)
V:I = 3:1 | V:I=2:1 | |
crtBI (28.2 ng/μl) | 3μl | 4μl |
luxPR (27.2 ng/μl) | 6μl | 6μl |
T DNA ligase | 1.5μl | 1.μl |
ligase buffer | 1.5μl | 1.5μl |
Water | 8μl | 7μl |
crtEBI | 3μl |
luxPR | 6μl |
buffer | 1.5μl |
ligase | 1.2μl |
water | 3.3μl |
DNA | 12μl |
EcoRI | 1μl |
Buffer | 2μl |
H2O | 5μl |
DNA | 10μl |
EcoRI | 1μl |
Buffer [Neb buffer EcoRI] | 2μl |
H2O | 7μl |
LuxPR | 17μl |
Spe I | 1μl |
Buffer (Cutsmart) | 2μl |
CrtEBI | 17μl |
Xba I | 1μl |
Buffer (Cutsmart) | 2μl |
LuxPR (128 ng/μl) | 3μl |
Buffer (EcoRI Buffer) | 2μl |
EcoRI | 1μl |
N.F./ dd H2O | 14μl |
CrtEBI (140 ng/μl ) | 3μl |
Buffer (2:1) | 2μl |
Xba I | 1μl |
N.F. H2O | 14μl |
DNA | 5μl |
EcoRI | 0.5μl |
Pst I | 0.5μl |
Dpa I | 0.5μl |
Buffer (2:1) | 2μl |
N.F. H2O | 11.5μl |
DNA (LuxPR) | 20μl |
Spe I | 1μl |
Buffer (Cutsmart) | 2μl |
DNA (CrtEBI) | 20μl |
Pst I | 1μl |
Buffer (3:1) | 2μl |
LuxPR | 3μl |
CrtEBI | 8μl |
Backbone | 5μl |
Buffer | 2μl |
Ligase (T4) | 1μl |
LuxPR | 6μl |
CrtEBI | 5μl |
Backbone | 3μl |
Buffer | 2μl |
Ligase (T4) | 1μl |
Water (T4) | 3μl |
17th August
DNA (68 ng) | 7μl |
Buffer (2:1) | 2μl |
EcoRI | 0.5μl |
Pst I | 0.5μl |
H2O | 10μl |
DNA | 7μl |
Buffer (CS) | 2μl |
Xba I | 0.5μl |
Spe I | 0.5μl |
H2O | 10μl |
DNA | 7μl |
Buffer (3:1) | 2μl |
Pst I | 1μl |
H2O | 10μl |
Total | 20μl |
3rd September
LuxR | 7μl |
EcoRI | 1μl |
Buffer (EcoRI) | 2μl |
dd H2O | 10μl |
CrtEBI | 10μl |
Xba I | 1μl |
Pst I | 2μl |
dBuffer (3:1) | 10μl |
dd H2O | 10μl |
DNA | 7μl |
Buffer (CS) | 2μl |
Xba I | 0.5μl |
Spe I | 0.5μl |
H2O | 10μl |
DNA | 7μl |
Buffer (3:1) | 2μl |
Pst I | 1μl |
H2O | 10μl |
Total | 20μl |
8th September
DNA | 13ul |
Spe 1 | 1µl |
Pst 1 | 1µl |
Buffer | 2ul |
dd H2O | 3ul |
9th September
DNA(7b) | 8µl |
Buffer (EcoR1) | 2µl |
dd H20 | 9µl |
DNA (CrtEBI) | 10µl |
Buffer (EcorR1) | 2µl |
dd H2O | 7µl |
DNA (LuxR) | 10µl |
Buffer (EcorR1) | 2µl |
EcorR1 | 1µl |
dd H2O | 7µl |
10th September
(X/P) CrtEBI (3.3) | 7µl |
(S/P) LuxPR (2.1) | 5µl |
T4 Ligase | 1µl |
Ligase Buffer | 2µl |
(X/P) CrtEBI (3.3) | 5µl |
(S/P) Lux R (3.3) | 8µl |
T4 Ligase | 1µl |
Buffer | 2µl |