Difference between revisions of "Team:Toulouse/Experiments"

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  <div class="shout">
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      <div class="title">
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    <center> <h3>Experiments & Protocols</h3> </center>
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    </div>
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  <center><img src="https://static.igem.org/mediawiki/2015/6/67/TLSE_BG.png"></center>
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  <div class="title">
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      <a href="#main1"><h3>Protocols for varroa tests</h3></a>
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  <ul>
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/* Page Header
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        <li><a href="#part1">- Sampling of varroas</a></li>
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--------------------------------------------------------------------------------------------------------------- */
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        <li><a href="#part2">- Standardization of varroas and sampling</a></li>
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#pageintro{padding:180px 0 0 0;}
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        <li><a href="#part3">- Attraction test on varroas</a></li>
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#pageintro .title{margin:0 0 50px 0;}
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        <li><a href="#part4">- Mortality test on varroas</a></li>
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      </ul>
 +
    </div>
 +
</center>
 +
 +
  <div class="title">
 +
      <a href="#main1"><h3>Transformation Protocol: RbCl method</h3></a>
 +
    </div>
 +
 +
<center> 
 +
    <div id="breadcrumb" class="clear" style="float: center;" >
 +
  <ul>
 +
        <li><a href="#part1">- Media and solution</a></li>
 +
        <li><a href="#part2">- Competent Cells</a></li>
 +
        <li><a href="#part3">- Miniprep</a></li>
 +
        <li><a href="#part4">- Transformation</a></li>
 +
      </ul>
 +
    </div>
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</center>
 +
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  <div class="title">
 +
      <a href="#main2"><h3>Cloning</h3></a>
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    </div>
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<center> 
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    <div id="breadcrumb" class="clear" style="float: center;" >
 +
  <ul>
 +
        <li><a href="#part6">- Digestion</a></li>
 +
        <li><a href="#part7">- Gel extraction</a></li>
 +
        <li><a href="#part8">- Ligation</a></li>
 +
<li><a href="#part9">- Transformation</a></li>
 +
      </ul>
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    </div>
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<hr style="width:66%;height:1px;border:none;color:rgba(29, 5, 79, 1);background-color:rgba(29, 5, 79, 1); z-index:50; position:relative;">
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</center>
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<div class="group center">
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<div class="title" >  
 +
<h3>Protocols for varroa tests</h3>
 +
</div>
 +
</div>
 +
<div class="group">
 +
<div class="subtitle" >  
 +
<h3>Sampling of varroa</h3>
 +
</div>
 +
</div>
 +
<div class="group" style="padding-top:10px">
 +
<div class="one_quarter first" >
 +
<div class="subtitle" style="margin-bottom:0px;">  
 +
<h3 style="font-size:20px;">Materials</h3>
 +
</div>
 +
<ul align="justify" style="font-size:15px;">
 +
<li>Bee hive </li>
 +
<li>Beekeeper suit</li>
 +
<li>Gloves</li>
 +
<li>Smoker</li>
 +
<li>Dry twigs</li>
 +
<li>Tweezers</li>
 +
<li>Big brush</li>
 +
<li>Small brush</li>
 +
<li>Petri dishes <br> Ø x h = 35 x 15 mm</li>
 +
</div>
  
 +
<div class="three_quarter">
 +
<div class="subtitle" style="margin-bottom:0px;">  
 +
<h3 style="font-size:20px;">Methods</h3>
 +
</div>
 +
<ol align="justify" style="font-size:15px;">
 +
<li>Slip beekeeper suit and gloves on and go to bee hive</li>
 +
<li>Fire dry twigs in smoker</li>
 +
<li>Open bee hive and activate smoker to get bees inside the hive</li>
 +
<li>Take a frame out the hive and remove bees with big brush and smoker</li>
 +
<li>Close bee hive</li>
 +
<li>In the lab, put the frame on a table against the wall</li>
 +
<li>With tweezer drill hole into one beehive cell</li>
 +
<li>Remove larvae and look for varroas on larvae and on beehive cell</li>
 +
<li>If there are varroas, take them with a small brush and put them on Petri dishes</li>
 +
<li>Make sure there are two or three larvae on Petri dishes in order to allow survival of varroas</li>
 +
<li>Start again step 7 to 9 until you have enough varroas</li>
 +
</ol>
 +
</div>
 +
</div>
  
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<h3>Standardization of varroas and sampling</h3>
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 +
<div class="group center">
 +
<p align="justify" style="font-size:15px;">When we take varroas directly from frame, as it is described in
 +
protocol “Sampling Varroas”, we have varroas in different phases. In order to have varroas
 +
in the same phase it is necessary to add one step and it is important for reproducibility of
 +
experiments. With this method we place varroas on adult bees so all varroas will be in phoretic phase.</p>
 +
</div>
 +
<div class="group" style="padding-top:10px">
 +
<div class="one_quarter first">
 +
<div class="subtitle" style="margin-bottom:0px;font-size:20px">  
 +
<h3 style="font-size:20px;">Materials</h3>
 +
</div>
 +
<ul align="justify" style="font-size:15px;">
 +
<li>Bees in box with aeration and glucose </li>
 +
<li>Varroas from protocol “Sampling varroas”</li>
 +
<li>Gas cylinder of CO<SUB>2</SUB></li>
 +
<li>Small brush </li>
 +
<li>Tweezers</li>
 +
<li>Petri dishes <br> Ø x h = 35 x 15 mm</li>
 +
</div>
  
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<div class="subtitle" style="margin-bottom:0px;font-size:20px">  
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<h3 style="font-size:20px;">Methods</h3>
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<li>With small brush take varroas from Petri dish and put them on bees in box through aeration holes</li>
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<li>Place the box in a 35°C incubator overnight. Make sure you have a bowl with water in order to have enough humidity in incubator</li>
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.clients ul li *:hover{opacity:1;}
+
<li>Take the box out of incubator</li>
 +
<li>Add CO<SUB>2</SUB> from gas cylinder into the box until all bees fall down</li>
 +
<li>Open the box, take a bee with tweezer and look for varroas</li>
 +
<li>When you find a varroa take him with small brush and replace bee in the box</li>
 +
<li>Start again step 5 and 6 until you have enough varroas</li>
 +
</ol>
 +
</div>
 +
</div>
  
 +
<div class="group">
 +
<div class="subtitle" >  
 +
<h3>Attraction test on varroas</h3>
 +
</div>
 +
</div>
 +
<div class="group center">
 +
<p align="justify" style="font-size:15px;">In order to test the attraction effect of butyric acid
 +
on varroas an Y test was used, as it is showed with diagram below. Where we place varroas for the
 +
test we choose glass pipe because on plastic they could load themselves with electrostatics and die.
 +
For butyric acid concentration we choose 4% (V/V) because this is the concentration used in the patent we see, see “Attribution” part. </p>
 +
</div>
  
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<div class="subtitle" style="margin-bottom:0px;font-size:20px">  
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<h3 style="font-size:20px;">Materials</h3>
 +
</div>
 +
<ul align="justify" style="font-size:15px;">
 +
 +
<li>Pump wich expels air</li>
 +
<li>15 mL Flacon tube</li>
 +
<li>Plastic pipe <br> Ø = 10mm</li>
 +
<li>Glass T pipe <br> Ø = 10mm, made by a glassworker</li>
 +
<li>Plastic separator</li>
 +
<li>Carded cotton</li>
 +
<li>Absorbent cotton</li>
 +
<li>5mL 4% (V/V) Butyric acid</li>
 +
<li>5mL Water</li>
 +
<li>Standardized varroas <br></li>
 +
</div>
  
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<div class="subtitle" style="margin-bottom:0px;font-size:20px">
 +
<h3 style="font-size:20px;">Methods</h3>
 +
</div>
 +
<ol align="justify" style="font-size:15px;">
 +
<li>Put a cotton on Petri dish and add 400µL of one acid formic solution</li>
 +
<li>Place three varroas on this Petri dish and close it</li>
 +
<li>Start again step1 and 2 for each formic acid solution and water</li>
 +
<li>Each 30 minutes check if varroas are alive. To do that :</li>
 +
<li>When varroa heads for one side of Glass T tube and covers more than 2 cm test is over and we write down the side choosen by varroa (Butyric acid or Water)</li>
 +
<li>Two tests can be made in the same time thanks to the plastic separator</li>
 +
</ol>
 +
</div>
 +
</div>
  
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/* Sidebar */
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<div class="group">
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.container .sidebar{}
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<div class="subtitle" >  
 +
<h3>Mortality test on varroas</h3>
 +
</div>
 +
</div>
 +
<div class="group center">
 +
<p align="justify" style="font-size:15px;">To test toxicity of formic acid
 +
on varroas we had to choose which concentrations we use. For that we based
 +
our thoughts on present treatments. When beekeepers use formic acid for long
 +
treatment they place a diffuser at the top of the hive and formic acid concentration
 +
was assessed at 200ppm<a target="_blank" href="http://www.agroscope.admin.ch/imkerei/00316/00329/02079/index.html?lang=en"> <SUP>1</SUP> </a>on average that is equivalent to 7,826 mmol.m<SUP>-3</SUP>.
 +
As gas concentration is difficult to evaluate we calculate the liquid concentration balance 
 +
thanks to the ideal gas law and the Henry’s law. To simplify calculation we noted down formic acid A.
 +
</p>
 +
</div>
  
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<div class="group center">
 +
<p style="font-size:15px;">
 +
$$ P\cdot V = n\cdot R\cdot T, \textrm{ideal gaz law} $$
 +
<!-- P. V = n.R.T, ideal gaz law-->
 +
$$ P_A = C_A\cdot R\cdot T = 7,826\cdot10^{-3}\times8,314\times293=19,964 Pa $$
 +
</p>
 +
</div>
 +
<ul style="font-size:15px;">
 +
<li>P<SUB>A</SUB>: partial pressure of A in Pa</li>
 +
<li>C<SUB>A</SUB>: Concentration of A in air in mol.m<SUP>-3</SUP></li>
 +
<li>R: perfect gaz constant = 8,314 J.mol<SUP>-1</SUP>.K<SUP>-1</SUP></li>
 +
<li>T: temperature in <SUP>°</SUP>K </li>
 +
</ul>
 +
<br>
 +
<div class="group center" style="padding-top:10px;">
 +
<p style="font-size:15px;">
 +
<!-- P_A = C_A.R.T = 7,826.10^3 x 8,314 x 293 = 19,964 Pa -->
 +
$$ P_A = H_A\cdot C_{A,eq}, \textrm{Henry's law} $$
 +
<!-- P_A = H_A . C_{A,eq}, Henry's law -->
 +
$$ C_{A,eq} = \frac{19,964}{0,019} = 1,019mol.L^{-1}$$
 +
</p>
 +
</div>
 +
<ul style="font-size:15px;">
 +
<li>C<SUB>A,eq</SUB>: equivalent concentration in liquid in mol.L<SUP>-1</SUP></li>
 +
<li>H<SUB>A</SUB>: Henry's constant = 0,019 Pa.m<SUP>3</SUP>mol<SUP>-1</SUP></li>
 +
</ul>
  
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<div class="subtitle" style="margin-bottom:0px;font-size:20px">  
 +
<h3 style="font-size:20px;">Materials</h3>
 +
</div>
 +
<ul align="justify" style="font-size:15px;">
 +
<li>Petri dishes <br> Ø x h = 35 x 15 mm</li>
 +
<li>Varroas form “Sampling varroas”</li>
 +
<li>Cotton</li>
 +
<li>Acid formic solutions : </li>
 +
<ul align="justify" style="font-size:15px;">
 +
<li>2 mol.L<SUP>-1</SUP></li>
 +
<li>10 mmol.L<SUP>-1</SUP></li>
 +
<li>1 mmol.L<SUP>-1</SUP></li>
 +
<li>500 µmol.L<SUP>-1</SUP></li>
 +
<li>50 µmol.L<SUP>-1</SUP></li>
 +
</ul>
 +
<li>Water</li>
 +
</div>
  
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#footer .shout{margin:0 0 80px 0; padding:0;}
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<div class="subtitle" style="margin-bottom:0px;font-size:20px">
 +
<h3 style="font-size:20px;">Methods</h3>
 +
</div>
 +
<ol align="justify" style="font-size:15px;">
 +
<li>Put a cotton on Petri dish and add 400µL of one acid formic solution</li>
 +
<li>Place three varroas on this Petri dish and close it</li>
 +
<li>Start again step1 and 2 for each formic acid solution and water</li>
 +
<li>Each 30 minutes check if varroas are alive. To do that : </li>
 +
<ol align="justify" style="font-size:15px;">
 +
<li value="5">Tap on Petri dish and see if varroa move. If yes varroa is alive, if not see below</li>
 +
<li>Look at binocular magnifier if varroas move their feets. If yes varroa is alive, if not varroa is dead <br>
 +
Remark: Varroa stop moving approximately one or two hours before it die </li>
 +
</li>
 +
</ol>
 +
</ol>
 +
</div>
 +
</div>
  
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<br>
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<center><hr style="width:66%;height:1px;border:none;color:rgba(29, 5, 79, 1);background-color:rgba(29, 5, 79, 1); z-index:50; position:relative;"></center>
 +
<div class="group center">
 +
<div class="title" >  
 +
<h3>Transformation Protocol: RbCl method</h3>
 +
</div>
 +
</div>
 +
<div class="group">
 +
<div class="subtitle">
 +
    <h3> Media and solution</h3>  <!-- MEDIA AND SOLUTION -->
 +
    </div>
 +
</div>
 +
<div class="group center">
 +
<table class="df">
 +
        <thead>
 +
          <tr>
 +
            <th>YETM 500mL</th>
 +
            <th>TFB1 200mL</th>
 +
            <th>TFB2 200mL</th>
 +
          </tr>
 +
        </thead>
 +
        <tbody>
 +
          <tr>
 +
            <td><!-- YETM 500mL -->
 +
<ul style="font-size:15px;"align="left">
 +
<li> 2.5g Yeast Extract </li>
 +
<li> 10g Tryptone </li>
 +
<li> 5g MgSO4.7H2O </li>
 +
<li> Adjust pH to 7.5 with KOH </li>
 +
<li> <b>For Plates</b>: add 7.5g of Agar </li>
 +
</ul>
 +
</td><!-- YETM 500mL END -->
 +
 +
<td><!-- TFB1 200mL -->
 +
<ul style="font-size:15px;" align="left">
 +
<li>  0.59g KOAc </li>
 +
<li> 2.42g RbCl </li>
 +
<li> 0.29g CaCl2.2H2O </li>
 +
<li> 1.98g MnCl2.4H2O </li>
 +
<li> Adjust to pH 5.8 with 0.2 M acetic acid </li>
 +
<li> Add dH2O to 200mL </li>
 +
<li> Filter sterilize </li>
 +
<li> Store refrigerated at 4°C </li>
 +
</ul>
 +
</td><!-- TFB1 200mL END -->
 +
 +
<td><!-- TFB2 200mL -->
 +
<ul style="font-size:15px;"align="left">
 +
<li> 0.42g MOPS </li>
 +
<li> 2.21g CaCl2.2H20 </li>
 +
<li> 0.24g RbCl </li>
 +
<li> 30g Glycerol </li>
 +
<li> Adjust to pH 6.5 with KOH </li>
 +
<li> Add dH2O to 200mL </li>
 +
<li> Filter sterilize </li>
 +
<li> Store refrigerated at 4°C </li>
 +
</ul>
 +
</td><!-- TFB2 200mL END -->
 +
          </tr>
 +
        </tbody>
 +
      </table>
 +
</div> <!-- MEDIA AND SOLUTION END -->
  
  
−
/* Copyright
+
<div class="subtitle">
−
---------------------------------------------------------------------------------------------------------------*/
+
    <h3> Preparation of Competent Cells </h3>  <!-- COMPETENT CELLS -->
−
#copyright{padding:30px 0;}
+
    </div>
−
#copyright p{margin:0; padding:0;}
+
<div class="group">
 +
<ul align="justify" style="font-size:15px;">
 +
<li>1. Streak cells froms frozen stock onto YETM plate. Incubate overnight at 37°C </li>
 +
<li>2. Pick a single fresh colony to inoculate 5mL of YETM medium. Grow over night at 37°C.</li>
 +
<li><b>Do not vortex cells at any time after this point in the procedure</b></li>
 +
<li>3. Dilute 1mL of culture into 50mL YETM medium prewarmed to 37°C</li>
 +
<ul align="justify" style="font-size:15px;">
 +
<li> Grow at 37°C for 2hr with agitation </li>
 +
<li> Volumes can be scaled up 5X and all of the 5mL overnight culture can be used </li>
 +
</ul>
 +
<li> 4. Transfer culture to sterile 50 mL tube. Chill on ice/water 10-15 minutes </li>
 +
<li> 5. Centrifuge for 10 minutes at 2000rpm at 4°C. Immediately aspirate off all of the supernatant </li>
 +
<li> <b> Do not allow cells to warm above 4°C at any time in this procedure </b> </li>
 +
<li> 6. Resuspend cells in 10mL of ice-cold TFB1 with gentle re-pipetting. Use chilled glass or plastic pipette </li>
 +
<li> 7. Incubate cells on ice for 5min </li>
 +
<li> 8. Repeat step 5</li>
 +
<li> 9. Resuspend cells in 2mL of ice-cold TFB2 with <b>gentle</b> re-pipetting. Use micropipet tip (plastic)</li>
 +
<li> 10. Incubate cells on ice for 15 minutes </li>
 +
<li> Cells may be used for transformation or frozen </li>
 +
<ul align="justify" style="font-size:15px;">
 +
<li> To freeze: aliquot cell in 200μL volumes into prechilled 0.5mL microfuge tube (on ice) </li>
 +
<li> Freeze immediately in liquid nitrogen </li>
 +
<li> Store cells frozen at -80°C </li>
 +
</ul>
 +
</ul>
 +
</div> <!-- COMPETENT CELLS END-->
 +
 +
<div class="subtitle">
 +
    <h3> Transformation of Competent Cells </h3>  <!-- COMPETENT CELLS -->
 +
</div>
 +
 +
<div class="group">
  
 +
<ul align="justify" style="font-size:15px;">
 +
<li>1. If starting with frozen competent cells, warm tube/cells by gently twirling between your fingers until just thawed. <br>Immedately place on ice for about 5 minutes </li>
 +
<li>2. Add to 1,5mL eppendorff on ice: 2-3μL iGEM plate or 1μL plasmid or 10μL ligation.</li>
 +
<li>3. Add 100μL of competent cells and mix by gentle re-pipetting</li>
 +
<li> 4. Incubate cells on ice for 20-30 minutes </li>
 +
<li> 5. Heat shock the cells exactly 90 seconds at 42°C </li>
 +
<li> 6. Return cells on ice for 2 minutes </li>
 +
<li> 7. Add 1mL of YETM medium. Incubate at 37°C for 45-60 minutes with slow gentle shaking </li>
 +
<li> 8. Plate 0.1-0.2 mL of transformed cells on LB-plate containing the appropriate antibiotic. Incubate  overnight at 37°C</li>
 +
</ul>
 +
</div> <!-- COMPETENT CELLS END -->
  
−
/* Back to Top
 
−
---------------------------------------------------------------------------------------------------------------*/
 
  
 +
<div class="subtitle">
 +
    <h3> Minipreps </h3>  <!-- MINIPREPS -->
 +
</div>
 +
 +
<div class="group">
  
−
/* Transition Fade
+
<ul align="justify" style="font-size:15px;">
−
--------------------------------------------------------------------------------------------------------------- */
+
<li>1. Resuspend 4 to 12 colonies from the plate and name each colony taken on the tubes and on the plate (A, B, C, …) </li>
−
.row0, .faico a, #mainav *, .shout-content, .overlay ::before, .overlay ::after, .boxedicon a, .clients ul li *, }
+
<li>2. Resuspend one colony per culture tube in 5 mL of LB medium with antibiotic</li>
 +
<li>3. Let the culture grow overnight at 37°C in a shaking incubator</li>
 +
<li> 4. Use the QIAprep spin Miniprep Kit for each culture tube. The last step consisting in the elution of the DNA is made with elution buffer or water at 55°C </li>
 +
<li> 5. Keep the tubes at -20°C</li>
 +
</div> <!-- MINIPREP END -->
 +
<br>
 +
<center><hr style="width:66%;height:1px;border:none;color:rgba(29, 5, 79, 1);background-color:rgba(29, 5, 79, 1); z-index:50; position:relative;"></center>
 +
<div class="group center">
 +
 +
<center><div class="title" >  
 +
<h3 >Cloning</h3><!-- CLONING -->
 +
</div></center>
 +
</div>
 +
<div class="subtitle" >  
 +
<h3>First step: Digestion</h3>
 +
</div>
  
 +
<div class="group center">
 +
<div class="subtitle" >  
 +
<h3 style="font-size:20px;">Both parts have the same antibiotic resistance</h3>
 +
</div>
 +
</div>
  
−
/*-------------------------------------------------------------------------------------------------------------*/
+
<div class="group center">
−
/*-------------------------------------------------------------------------------------------------------------*/
+
<table class="df" style="font-size:15px;">
−
/*-------------------------------------------------------------------------------------------------------------*/
+
        <thead>
−
/*-------------------------------------------------------------------------------------------------------------*/
+
          <tr>
−
/*-------------------------------------------------------------------------------------------------------------*/
+
            <th>Vector</th>
 +
<th>Insert</th>
 +
          </tr>
 +
        </thead>
 +
        <tbody>
 +
 +
          <tr>
 +
            <td><p>1 µg of </p></td>
 +
            <td><p>10 µL of miniprep plasmid</p></td>
 +
       
 +
          </tr>
 +
          <tr>
 +
            <td><p>1 µL of each restriction enzymes</p></td>
 +
            <td><p>1 µL of each restriction enzymes</p></td>
 +
         
 +
          </tr>
 +
          <tr>
 +
            <td><p>2 µL of Green Buffer</p></td>
 +
            <td><p>2 µL of Green Buffer</p></td>
 +
           
 +
          </tr>
 +
          <tr>
 +
            <td><p>9 µL of Milli-Q water</p></td>
 +
            <td><p>4 µL of Milli-Q water</p></td>
 +
          </tr>
 +
  <tr>
 +
  <td colspan=2><p>Incubate 15 minutes at 37°C</p></td>
 +
  </tr>
 +
        </tbody>
 +
      </table>
 +
  </div>
 +
 
 +
 
 +
  <div class="group center">
 +
<center><div class="subtitle">  
 +
<h3 style="font-size:20px;">The two parts have a different antibiotic resistance</h3>
 +
</div></center>
 +
</div>
  
 +
<div class="group center">
 +
<table class="df" style="font-size:15px;">
 +
        <thead>
 +
          <tr>
 +
            <th>Both parts</th>
 +
          </tr>
 +
        </thead>
 +
        <tbody>
 +
          <tr>
 +
            <td><p>5 µL of miniprep plasmid</p></td>
 +
          </tr>
 +
          <tr>
 +
            <td><p>1 µL of each restriction enzymes</p></td>
 +
          </tr>
 +
          <tr>
 +
            <td><p>2 µL of Green Buffer</p></td>
 +
          </tr>
 +
          <tr>
 +
            <td><p>9 µL of Milli-Q water</p></td>
 +
          </tr>
 +
  <tr>
 +
  <td><p>Incubate 15 minutes at 37°C</p></td>
 +
  </tr>
 +
        </tbody>
 +
      </table>
 +
  </div>
 +
 
 +
<center><div class="subtitle" >  
 +
<h3 style="font-size:20px;">Migration and gel extraction</h3>
 +
</div></center> 
  
−
/* Navigation
+
<div class="group">
−
---------------------------------------------------------------------------------------------------------------*/
+
<ul align="justify" style="font-size:15px;">
−
nav ul, nav ol{margin:0; padding:0; list-style:none;}
+
<li>1. Prepare a 1% or 2% electrophoresis agarose gel with 0.5x TAE buffer </li>
 +
<li>2. Put 20 µL of sample + 6 µL of marker (1 kb for 1% gel and 100 pb for 2%) into the well</li>
 +
<li>3. Migration for 30 min at 100 V or 1 hour at 50 V</li>
 +
<li> 4. Bathe 10 minutes in BET</li>
 +
<li> 5. Wash in water for 5 minutes </li>
 +
<li> 6. The gel extraction is realized thanks to the QIAGEN Gel Extraction Kit</li>
 +
<br>
 +
<li><u> Two ways to inactivate the enzymes for the vector</u></li>
 +
<ul align="justify" style="font-size:15px;">
 +
<li> Use of DNA Clean up kit for the DNA fragment above 200 pb</li>
 +
<li> Heat inactivation at 95°C for 10 minutes </li>
 +
</ul>
 +
</ul>
 +
</div>
  
−
#mainav .drop::after, #mainav li li .drop::after, #breadcrumb li a::after, .sidebar nav a::after{position:absolute; font-family:"FontAwesome"; font-size:50px; line-height:10px;}
+
 
 +
 
 +
<div class="subtitle" >  
 +
<h3>Second step: Ligation</h3>
 +
</div> 
 +
 
 +
<div class="group center">
 +
<table class="df" style="font-size:15px;">
 +
        <thead>
 +
          <tr>
 +
            <th>Mix</th>
 +
<th>Control</th>
 +
          </tr>
 +
        </thead>
 +
        <tbody>
 +
          <tr>
 +
            <td><p>10 µL of insert</p></td>
 +
<td><p>no insert</p></td>
 +
          </tr>
 +
          <tr>
 +
            <td><p>4 µL of vector</p></td>
 +
<td><p>4 µL of vector</p></td>
 +
          </tr>
 +
          <tr>
 +
            <td><p>2 µL of 10x T4 buffer</p></td>
 +
<td><p>2 µL of 10x T4 buffer</p></td>
 +
          </tr>
 +
          <tr>
 +
            <td><p>0.5 µL of T4 ligase</p></td>
 +
<td><p>0.5 µL of T4 ligase</p></td>
 +
          </tr>
 +
  <tr>
 +
  <td><p>3.5 µL of Milli-Q water</p></td>
 +
  <td><p>13.5 µL of Milli-Q water</p></td>
 +
  </tr>
 +
  <tr>
 +
  <td colspan=2><p>&nbsp;Incubate the ligation mix 15 minutes at room temperature (22°C)</p></td>
 +
  </tr>
 +
  <tr>
 +
  <td colspan=2><p>&nbsp;Keep the tubes in ice or at -20°C to prepare the transformation</p></td>
 +
  </tr>
 +
        </tbody>
 +
      </table>
 +
  </div>
 +
 
 +
<div class="subtitle" >  
 +
<h3>Third step: Ligation</h3>
 +
</div> 
  
−
/* Top Navigation */
+
<div class="group">
−
#mainav{margin-bottom:-1px; }
+
<ul align="justify" style="font-size:15px;">
−
#mainav ul{text-transform:uppercase;margin-top:-1px;}
+
<li>1. Take 10µL of the ligation mix for 100 µL of competent cells and use the Toulouse iGEM Team 2015 transformation protocol </li>
−
#mainav ul ul{z-index:9999; position:absolute; width:160px; text-align:left; text-transform:none;}
+
<li>2. Plate the solution on selective medium overnight at 37°C</li>
−
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+
</ul>
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+
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+
 
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+
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+
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+
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+
  
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+
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References
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+
  
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<!-- #######################        FOOTER        ######################################################################### -->
  
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+
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Revision as of 14:30, 2 September 2015

iGEM Toulouse 2015

Experiments & Protocols


Protocols for varroa tests

Transformation Protocol: RbCl method

Cloning

Protocols for varroa tests

Sampling of varroa

Materials

  • Bee hive
  • Beekeeper suit
  • Gloves
  • Smoker
  • Dry twigs
  • Tweezers
  • Big brush
  • Small brush
  • Petri dishes
    Ø x h = 35 x 15 mm

Methods

  1. Slip beekeeper suit and gloves on and go to bee hive
  2. Fire dry twigs in smoker
  3. Open bee hive and activate smoker to get bees inside the hive
  4. Take a frame out the hive and remove bees with big brush and smoker
  5. Close bee hive
  6. In the lab, put the frame on a table against the wall
  7. With tweezer drill hole into one beehive cell
  8. Remove larvae and look for varroas on larvae and on beehive cell
  9. If there are varroas, take them with a small brush and put them on Petri dishes
  10. Make sure there are two or three larvae on Petri dishes in order to allow survival of varroas
  11. Start again step 7 to 9 until you have enough varroas

Standardization of varroas and sampling

When we take varroas directly from frame, as it is described in protocol “Sampling Varroas”, we have varroas in different phases. In order to have varroas in the same phase it is necessary to add one step and it is important for reproducibility of experiments. With this method we place varroas on adult bees so all varroas will be in phoretic phase.

Materials

  • Bees in box with aeration and glucose
  • Varroas from protocol “Sampling varroas”
  • Gas cylinder of CO2
  • Small brush
  • Tweezers
  • Petri dishes
    Ø x h = 35 x 15 mm

Methods

  1. With small brush take varroas from Petri dish and put them on bees in box through aeration holes
  2. Place the box in a 35°C incubator overnight. Make sure you have a bowl with water in order to have enough humidity in incubator
  3. Take the box out of incubator
  4. Add CO2 from gas cylinder into the box until all bees fall down
  5. Open the box, take a bee with tweezer and look for varroas
  6. When you find a varroa take him with small brush and replace bee in the box
  7. Start again step 5 and 6 until you have enough varroas

Attraction test on varroas

In order to test the attraction effect of butyric acid on varroas an Y test was used, as it is showed with diagram below. Where we place varroas for the test we choose glass pipe because on plastic they could load themselves with electrostatics and die. For butyric acid concentration we choose 4% (V/V) because this is the concentration used in the patent we see, see “Attribution” part.

Materials

  • Pump wich expels air
  • 15 mL Flacon tube
  • Plastic pipe
    Ø = 10mm
  • Glass T pipe
    Ø = 10mm, made by a glassworker
  • Plastic separator
  • Carded cotton
  • Absorbent cotton
  • 5mL 4% (V/V) Butyric acid
  • 5mL Water
  • Standardized varroas

Methods

  1. Put a cotton on Petri dish and add 400µL of one acid formic solution
  2. Place three varroas on this Petri dish and close it
  3. Start again step1 and 2 for each formic acid solution and water
  4. Each 30 minutes check if varroas are alive. To do that :
  5. When varroa heads for one side of Glass T tube and covers more than 2 cm test is over and we write down the side choosen by varroa (Butyric acid or Water)
  6. Two tests can be made in the same time thanks to the plastic separator

Mortality test on varroas

To test toxicity of formic acid on varroas we had to choose which concentrations we use. For that we based our thoughts on present treatments. When beekeepers use formic acid for long treatment they place a diffuser at the top of the hive and formic acid concentration was assessed at 200ppm 1 on average that is equivalent to 7,826 mmol.m-3. As gas concentration is difficult to evaluate we calculate the liquid concentration balance thanks to the ideal gas law and the Henry’s law. To simplify calculation we noted down formic acid A.

$$ P\cdot V = n\cdot R\cdot T, \textrm{ideal gaz law} $$ $$ P_A = C_A\cdot R\cdot T = 7,826\cdot10^{-3}\times8,314\times293=19,964 Pa $$

  • PA: partial pressure of A in Pa
  • CA: Concentration of A in air in mol.m-3
  • R: perfect gaz constant = 8,314 J.mol-1.K-1
  • T: temperature in °K

$$ P_A = H_A\cdot C_{A,eq}, \textrm{Henry's law} $$ $$ C_{A,eq} = \frac{19,964}{0,019} = 1,019mol.L^{-1}$$

  • CA,eq: equivalent concentration in liquid in mol.L-1
  • HA: Henry's constant = 0,019 Pa.m3mol-1

Materials

  • Petri dishes
    Ø x h = 35 x 15 mm
  • Varroas form “Sampling varroas”
  • Cotton
  • Acid formic solutions :
    • 2 mol.L-1
    • 10 mmol.L-1
    • 1 mmol.L-1
    • 500 µmol.L-1
    • 50 µmol.L-1
  • Water

Methods

  1. Put a cotton on Petri dish and add 400µL of one acid formic solution
  2. Place three varroas on this Petri dish and close it
  3. Start again step1 and 2 for each formic acid solution and water
  4. Each 30 minutes check if varroas are alive. To do that :
    1. Tap on Petri dish and see if varroa move. If yes varroa is alive, if not see below
    2. Look at binocular magnifier if varroas move their feets. If yes varroa is alive, if not varroa is dead
      Remark: Varroa stop moving approximately one or two hours before it die

Transformation Protocol: RbCl method

Media and solution

YETM 500mL TFB1 200mL TFB2 200mL
  • 2.5g Yeast Extract
  • 10g Tryptone
  • 5g MgSO4.7H2O
  • Adjust pH to 7.5 with KOH
  • For Plates: add 7.5g of Agar
  • 0.59g KOAc
  • 2.42g RbCl
  • 0.29g CaCl2.2H2O
  • 1.98g MnCl2.4H2O
  • Adjust to pH 5.8 with 0.2 M acetic acid
  • Add dH2O to 200mL
  • Filter sterilize
  • Store refrigerated at 4°C
  • 0.42g MOPS
  • 2.21g CaCl2.2H20
  • 0.24g RbCl
  • 30g Glycerol
  • Adjust to pH 6.5 with KOH
  • Add dH2O to 200mL
  • Filter sterilize
  • Store refrigerated at 4°C

Preparation of Competent Cells

  • 1. Streak cells froms frozen stock onto YETM plate. Incubate overnight at 37°C
  • 2. Pick a single fresh colony to inoculate 5mL of YETM medium. Grow over night at 37°C.
  • Do not vortex cells at any time after this point in the procedure
  • 3. Dilute 1mL of culture into 50mL YETM medium prewarmed to 37°C
    • Grow at 37°C for 2hr with agitation
    • Volumes can be scaled up 5X and all of the 5mL overnight culture can be used
  • 4. Transfer culture to sterile 50 mL tube. Chill on ice/water 10-15 minutes
  • 5. Centrifuge for 10 minutes at 2000rpm at 4°C. Immediately aspirate off all of the supernatant
  • Do not allow cells to warm above 4°C at any time in this procedure
  • 6. Resuspend cells in 10mL of ice-cold TFB1 with gentle re-pipetting. Use chilled glass or plastic pipette
  • 7. Incubate cells on ice for 5min
  • 8. Repeat step 5
  • 9. Resuspend cells in 2mL of ice-cold TFB2 with gentle re-pipetting. Use micropipet tip (plastic)
  • 10. Incubate cells on ice for 15 minutes
  • Cells may be used for transformation or frozen
    • To freeze: aliquot cell in 200μL volumes into prechilled 0.5mL microfuge tube (on ice)
    • Freeze immediately in liquid nitrogen
    • Store cells frozen at -80°C

Transformation of Competent Cells

  • 1. If starting with frozen competent cells, warm tube/cells by gently twirling between your fingers until just thawed.
    Immedately place on ice for about 5 minutes
  • 2. Add to 1,5mL eppendorff on ice: 2-3μL iGEM plate or 1μL plasmid or 10μL ligation.
  • 3. Add 100μL of competent cells and mix by gentle re-pipetting
  • 4. Incubate cells on ice for 20-30 minutes
  • 5. Heat shock the cells exactly 90 seconds at 42°C
  • 6. Return cells on ice for 2 minutes
  • 7. Add 1mL of YETM medium. Incubate at 37°C for 45-60 minutes with slow gentle shaking
  • 8. Plate 0.1-0.2 mL of transformed cells on LB-plate containing the appropriate antibiotic. Incubate overnight at 37°C

Minipreps

  • 1. Resuspend 4 to 12 colonies from the plate and name each colony taken on the tubes and on the plate (A, B, C, …)
  • 2. Resuspend one colony per culture tube in 5 mL of LB medium with antibiotic
  • 3. Let the culture grow overnight at 37°C in a shaking incubator
  • 4. Use the QIAprep spin Miniprep Kit for each culture tube. The last step consisting in the elution of the DNA is made with elution buffer or water at 55°C
  • 5. Keep the tubes at -20°C

Cloning

First step: Digestion

Both parts have the same antibiotic resistance

Vector Insert

1 µg of

10 µL of miniprep plasmid

1 µL of each restriction enzymes

1 µL of each restriction enzymes

2 µL of Green Buffer

2 µL of Green Buffer

9 µL of Milli-Q water

4 µL of Milli-Q water

Incubate 15 minutes at 37°C

The two parts have a different antibiotic resistance

Both parts

5 µL of miniprep plasmid

1 µL of each restriction enzymes

2 µL of Green Buffer

9 µL of Milli-Q water

Incubate 15 minutes at 37°C

Migration and gel extraction

  • 1. Prepare a 1% or 2% electrophoresis agarose gel with 0.5x TAE buffer
  • 2. Put 20 µL of sample + 6 µL of marker (1 kb for 1% gel and 100 pb for 2%) into the well
  • 3. Migration for 30 min at 100 V or 1 hour at 50 V
  • 4. Bathe 10 minutes in BET
  • 5. Wash in water for 5 minutes
  • 6. The gel extraction is realized thanks to the QIAGEN Gel Extraction Kit

  • Two ways to inactivate the enzymes for the vector
    • Use of DNA Clean up kit for the DNA fragment above 200 pb
    • Heat inactivation at 95°C for 10 minutes

Second step: Ligation

Mix Control

10 µL of insert

no insert

4 µL of vector

4 µL of vector

2 µL of 10x T4 buffer

2 µL of 10x T4 buffer

0.5 µL of T4 ligase

0.5 µL of T4 ligase

3.5 µL of Milli-Q water

13.5 µL of Milli-Q water

 Incubate the ligation mix 15 minutes at room temperature (22°C)

 Keep the tubes in ice or at -20°C to prepare the transformation

Third step: Ligation

  • 1. Take 10µL of the ligation mix for 100 µL of competent cells and use the Toulouse iGEM Team 2015 transformation protocol
  • 2. Plate the solution on selective medium overnight at 37°C

References