Difference between revisions of "Team:IIT Delhi/recipe"

 
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#banner {
 
#banner {
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background: url("https://static.igem.org/mediawiki/2017/8/8d/T--IIT_DELHI--banner_index.jpg") ;
+
background: url("https://static.igem.org/mediawiki/2017/8/80/T--IIT_Delhi--notebook.png") ;
  
 
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}
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.dropbtn2 {
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    font-size: 16px;   
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    border: none;
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    outline: none;
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    color: white;
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    padding: 15px 16px;
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    background-color: #000;
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    background-size: 100% 100%;
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    z-index: 101;
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}
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.navbar a:hover, .dropdown:hover .dropbtn {
 
.navbar a:hover, .dropdown:hover .dropbtn {
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<div class = "navbar ">
 
<div class = "navbar ">
 
 
−
                                                         <button class="dropbtn1"><a href="https://2017.igem.org/Team:IIT_Delhi">iGEM IIT Delhi</a></button>
+
                                                         <button class="dropbtn2"><a href="https://2017.igem.org/Team:IIT_Delhi">iGEM IIT Delhi</a></button>
−
<div class = "right_menu">
+
        <div class = "right_menu">
 
   <div class="dropdown">
 
   <div class="dropdown">
 
     <button class="dropbtn">Project  
 
     <button class="dropbtn">Project  
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     </button>
 
     </button>
 
     <div class="dropdown-content">
 
     <div class="dropdown-content">
−
       <a href="/Team:IIT_Delhi/Description">Description</a>
+
       <a href="/Team:IIT_Delhi/Description">Overview</a>
−
       <a href="/Team:IIT_Delhi/Results">Results</a>
+
       <a href="/Team:IIT_Delhi/Design">Squarewave Generator</a>
  
−
       <a href="/Team:IIT_Delhi/Interlab">Interlab</a>
+
       <a href="/Team:IIT_Delhi/InterLab">Interlab</a>
 
     </div>
 
     </div>
 
   </div>  
 
   </div>  
 
                                                         <div class="dropdown">
 
                                                         <div class="dropdown">
 +
    <button class="dropbtn">Results
 +
    <i class="fa fa-caret-down"></i>
 +
    </button>
 +
    <div class="dropdown-content">
 +
      <a href="/Team:IIT_Delhi/Circuit_Design">Circuit design and construction</a>
 +
      <a href="/Team:IIT_Delhi/Microfluidics">Microfluidics and Fluorescence</a>
 +
      <a href="/Team:IIT_Delhi/Photobleaching">Photobleaching</a>
 +
      <a href="/Team:IIT_Delhi/Promoter">Promoter strength</a>
 +
                                                      <a href="/Team:IIT_Delhi/Oscillations">Oscillations</a>
 +
                                                     
 +
    </div>
 +
  </div>
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                                                          <div class="dropdown">
 
     <button class="dropbtn">Parts  
 
     <button class="dropbtn">Parts  
 
     <i class="fa fa-caret-down"></i>
 
     <i class="fa fa-caret-down"></i>
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       <a href="/Team:IIT_Delhi/Improved_Part">Improved Parts</a>
 
       <a href="/Team:IIT_Delhi/Improved_Part">Improved Parts</a>
 
       <a href="/Team:IIT_Delhi/Part_Collection">Part Collection</a>
 
       <a href="/Team:IIT_Delhi/Part_Collection">Part Collection</a>
 +
                                                     
 
     </div>
 
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   <div class="dropdown">
 
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     <button class="dropbtn">Modeling
 
     <button class="dropbtn">Modeling
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     <div class="dropdown-content-big">
 
     <div class="dropdown-content-big">
 
       <a href="/Team:IIT_Delhi/Model">Overview</a>
 
       <a href="/Team:IIT_Delhi/Model">Overview</a>
−
       <a href="/Team:IIT_Delhi/Database">Simulation Database</a>
+
       <a href="/Team:IIT_Delhi/Write_Model">Writing a Model</a>
 +
                                                        <a href="/Team:IIT_Delhi/Deterministic_Model">Deterministic Model </a>
 +
                                                        <a href="/Team:IIT_Delhi/Stochastic_Model">Stochastic Model</a>
 +
                                                        <a href="/Team:IIT_Delhi/Bifurcation">Bifurcation and Squareness</a>
 +
                                                        <a href="/Team:IIT_Delhi/Resources">Resource sharing</a>
 
     </div>
 
     </div>
 
     </div>
 
     </div>
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       <a href="/Team:IIT_Delhi/Engagement">Public Engagement</a>
 
       <a href="/Team:IIT_Delhi/Engagement">Public Engagement</a>
 
       <a href="/Team:IIT_Delhi/Collaborations">Collaborations</a>
 
       <a href="/Team:IIT_Delhi/Collaborations">Collaborations</a>
 +
                                                        <a href="/Team:IIT_Delhi/Safety">Safety</a>
 
     </div>
 
     </div>
 
   </div>  
 
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                                                        <a href="/Team:IIT_Delhi/Collaborations">Overview</a>
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      <a href="/Team:IIT_Delhi/GMM_legislation">GMM Legislation</a>
 +
      <a href="/Team:IIT_Delhi/berlin">iGEM Berlin</a>
 +
      <a href="/Team:IIT_Delhi/mohali">Mentoring IISER Mohali</a>
 +
      <a href="/Team:IIT_Delhi/glasgow">iGEM Glasgow</a>
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     <button class="dropbtn">Notebook  
 
     <button class="dropbtn">Notebook  
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−
             <h2 class="h2font">LAB PROTOCOLS</h2>
+
             <h2 class="h2font">Recipes</h2>
  
 
             <p> &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 
             <p> &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
 
  </p>
 
  </p>
−
<h2 id="pfont">Accurate Experimenting for Accurate Results !!</h2>
+
<h2 id="pfont">A pinch of salt makes all the difference !!</h2>
 
           </header>
 
           </header>
−
           <button class="accordion back1" style="font-weight: bold;">Transformation</button>
+
           <button class="accordion back1" style="font-weight: bold;">Preparation of TAE buffer</button>
−
           <div class="panel ">
+
           <div class="panel">
−
           
+
          <div align="left" style="font-size:84%;">  
−
<!--<u><b><h4 id="pfont"  style="font-size: 90%;">Preparation of competent  cells:-</h4></b></u>-->
+
            &nbsp;&nbsp;&nbsp;&nbsp; 20x TAE buffer
−
<b id="pfont">Preparation of competent  cells:-</b>
+
          <ul>
−
<div align="left" style="font-size: 84%;">
+
          <li>80mL of 1M Tris solution
−
<ol ><left>
+
          <li>0.7264g of EDTANa2+ salt
−
<li>Dilute an overnight culture of E. coli 1:200 with LB broth.
+
          <li>2.284 mL glacial acetic acid
−
<li>Incubate at 37°C with shaking (at 200 rpm) until the cells reach early log phase (OD600 = 0.25-0.4).
+
          <li>distilled H2O 100mL
−
<li>We already have 1X TSS in 4 deg fridge(old). Use it without dilution or thawing. Keep it inside the icebox just after taking out from the fridge. <br>
+
          </ul>
−
OR <br>
+
<br>
−
(if the above 1X TSS is not available)<br>
+
−
While cells are growing, thaw 2X TSS on ice and dilute an appropriate amount 1:1 with sterile distilled water (100µl of diluted TSS will be needed for each ml of cells). Chill on ice.
+
−
<li> Place 2-ml aliquots of early log-phase cells into sterile 2-ml micro-centrifuge tubes and pellet the cells by centrifugation at 4°C  at 3000g for 10 min.(6-8 mins for taking part from Igem kit)
+
−
<li> Remove the supernatant and discard. Add 0.2 ml of the ice-cold 1X TSS and place the tubes on ice.
+
−
<li>Gently suspend the cells by pipetting.
+
−
<li> Proceed with the transformation protocol below (Step 2), or immediately freeze cells by immersion in liquid nitrogen or a dry ice/ethanol bath. Store the frozen cells at –70°C.
+
−
</left>
+
−
</ol>
+
 
</div>
 
</div>
−
 
+
</div>
−
<b id="pfont">Transformation:-</b>
+
          <button class="accordion back2" style="font-weight: bold;">Preparation of Ethidium Bromide(EtBr)</button>
 +
          <div class="panel">
 +
          <div align="left" style="font-size:84%;">
 +
          <ul>
 +
<li>For the preparation of ethidium bromide adds 1 g of ethidium bromide to 100 ml of H2O.
 +
<li>Stir on a magnetic stirrer for several hours to ensure that the dye has dissolved.
 +
<li> Wrap the container in aluminum foil or transfer the 10 mg/ml solution to a dark bottle and
 +
store at room temperature. </ul>
 
<br>
 
<br>
−
<div align="left" style="font-size: 84%;">
+
          </div>
−
<ol ><left>
+
          </div>
−
<li>Thaw frozen TSS-competent cells slowly on ice(if stored at -70°C).  
+
<button class="accordion back3" style="font-weight: bold;">Preparation of alkaline lysis solution I</button>
−
<li>Add 100 pg -200 ng (2.5 to 4 ul)(15ul for ligation product)of DNA to each tube of competent cells. <br>Note:Addition of more than 10ng of DNA may significantly decrease transformation efficiencies.  
+
          <div class="panel">
−
<li>Flick the tubes to mix the cells and DNA and incubate the cells on ice for 30 minutes. <br>
+
          <div align="left" style="font-size:84%;">  
 +
          <ul>
 +
          <li>50 mM glucose.
 +
          <li>25 mM Tris-Cl (pH 8.0).
 +
          <li>10 mM EDTA (pH 8.0).
 +
          </ul>
 +
          <ul>Prepare Solution I from standard stocks in batches of approx. 100 ml, sterilize by
 +
autoclaving and store at 4°C.</ul>
 +
<br>
 +
          </div>
 +
          </div>
 +
<button class="accordion back4" style="font-weight: bold;">Preparation of alkaline lysis solution II (freshly prepared at room temperature)</button>
 +
          <div class="panel">
 +
          <div align="left" style="font-size:84%;">
 +
          <ul>
 +
<li>0.2 N NaOH (freshly diluted from a 10 N stock).
 +
<li>(100ul SDS + 700ul mq + 200ul NaOH)
 +
1% (w/v) SDS.</ul>
 +
<br>
 +
          </div>
 +
</div>
 +
<button class="accordion back1" style="font-weight: bold;">Preparation of alkaline lysis solution III</button>
 +
          <div class="panel">
 +
          <div align="left" style="font-size:84%;">
 +
        <ul>
 +
        <li>5 M potassium acetate, 60.0 ml
 +
        <li>Glacial acetic acid, 11.5 ml
 +
        <li>H2O, 28.5 ml
 +
        </ul>
 +
          <ul>The resulting solution is 3 M with respect to potassium and 5 M with respect to acetate.
 +
Store the solution at 4°C and transfer it to an ice bucket just before use.</ul>
 +
<br>
 +
          </div>
  
−
<li>  Transfer the tubes to water bath/dry bath(42°C) for 90 seconds.
+
</div>
−
<li> Transfer the tubes to ice and incubate for an additional 10 minutes.
+
−
<li>Add 800 ul (total 1 mL)of LB broth and incubate the cells at 37°C for up to 1 hour with shaking (at 200 rpm).
+
  
−
<li> Centrifuge the cells at 3000g for ~ 6min (10 mins after ligation)at 4deg(in temperature control centrifuge).
+
    <br>  
−
   <li> Aspirate the tubes to leave the pellets with 1/4 broth .(keep ~300ul)
+
          <script>
−
    <li>Plate the cells on-to the appropriate selective or differential medium and incubate overnight at 37°C.Check the procedure for antibiotic.
+
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      <ol> <li>For Ampicillin: 12ul
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            <td align="center" ><b>Address</b></td>
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<center> <p  style="text-align: center; color:white; font-size: 80%;"><b>E-mail:</b> iitd.igem@gmail.com<br>Undergraduate Laboratory<br>Department of Biotechnology and Biochemical Engineering, IIT Delhi</p></center>
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Latest revision as of 22:22, 1 November 2017

iGEM IIT Delhi

Overview Squarewave Generator Interlab
Circuit design and construction Microfluidics and Fluorescence Photobleaching Promoter strength Oscillations
Basic Parts Composite Parts Improved Parts Part Collection
Overview Writing a Model Deterministic Model Stochastic Model Bifurcation and Squareness Resource sharing
Human Practices Integrated Practices Public Engagement Collaborations Safety
Overview GMM Legislation iGEM Berlin Mentoring IISER Mohali iGEM Glasgow
Lab Records Protocols Recipes Medal Criteria
The Team Attributions

Recipes

                                                                                                                                                                                                                 

A pinch of salt makes all the difference !!

     20x TAE buffer
  • 80mL of 1M Tris solution
  • 0.7264g of EDTANa2+ salt
  • 2.284 mL glacial acetic acid
  • distilled H2O 100mL

  • For the preparation of ethidium bromide adds 1 g of ethidium bromide to 100 ml of H2O.
  • Stir on a magnetic stirrer for several hours to ensure that the dye has dissolved.
  • Wrap the container in aluminum foil or transfer the 10 mg/ml solution to a dark bottle and store at room temperature.

  • 50 mM glucose.
  • 25 mM Tris-Cl (pH 8.0).
  • 10 mM EDTA (pH 8.0).
    Prepare Solution I from standard stocks in batches of approx. 100 ml, sterilize by autoclaving and store at 4°C.

  • 0.2 N NaOH (freshly diluted from a 10 N stock).
  • (100ul SDS + 700ul mq + 200ul NaOH) 1% (w/v) SDS.

  • 5 M potassium acetate, 60.0 ml
  • Glacial acetic acid, 11.5 ml
  • H2O, 28.5 ml
    The resulting solution is 3 M with respect to potassium and 5 M with respect to acetate. Store the solution at 4°C and transfer it to an ice bucket just before use.


Sponsored By
Contact Us Address

E-mail: iitd.igem@gmail.com
Undergraduate Laboratory
Department of Biotechnology and Biochemical Engineering, IIT Delhi