Difference between revisions of "Team:Toulouse/Experiments"

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    <center> <h3>Experiments & Protocols</h3> </center>
 
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      <a href="#main1"><h3>Protocols for varroa tests</h3></a>
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        <li><a href="#part1">- Sampling of varroas</a></li>
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/* Page Header
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        <li><a href="#part2">- Standardization of varroas and sampling</a></li>
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        <li><a href="#part3">- Attraction test on varroas</a></li>
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#pageintro{padding:180px 0 0 0;}
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        <li><a href="#part4">- Mortality test on varroas</a></li>
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      <a href="#main1"><h3>Transformation Protocol: RbCl method</h3></a>
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<center> 
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    <div id="breadcrumb" class="clear" style="float: center;" >
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  <ul>
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        <li><a href="#part1">- Media and solution</a></li>
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        <li><a href="#part2">- Competent Cells</a></li>
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        <li><a href="#part3">- Miniprep</a></li>
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        <li><a href="#part4">- Transformation</a></li>
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      <a href="#main2"><h3>Cloning</h3></a>
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    <div id="breadcrumb" class="clear" style="float: center;" >
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  <ul>
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        <li><a href="#part6">- Digestion</a></li>
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        <li><a href="#part7">- Gel extraction</a></li>
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        <li><a href="#part8">- Ligation</a></li>
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<li><a href="#part9">- Transformation</a></li>
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<div class="title" >  
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<h3>Protocols for varroa tests</h3>
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</div>
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</div>
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<div class="group">
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<div class="subtitle" >  
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<h3>Sampling of varroa</h3>
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</div>
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</div>
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<div class="one_quarter first" >
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<div class="subtitle" style="margin-bottom:0px;">  
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<h3 style="font-size:20px;">Materials</h3>
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</div>
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<ul align="justify" style="font-size:15px;">
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<li>Bee hive </li>
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<li>Beekeeper suit</li>
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<li>Gloves</li>
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<li>Smoker</li>
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<li>Dry twigs</li>
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<li>Tweezers</li>
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<li>Big brush</li>
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<li>Small brush</li>
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<li>Petri dishes <br> Ø x h = 35 x 15 mm</li>
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</div>
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<div class="three_quarter">
 
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<div class="subtitle" style="margin-bottom:0px;">  
 
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<h3 style="font-size:20px;">Methods</h3>
 
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</div>
 
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<ol align="justify" style="font-size:15px;">
 
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<li>Slip beekeeper suit and gloves on and go to bee hive</li>
 
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<li>Fire dry twigs in smoker</li>
 
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<li>Open bee hive and activate smoker to get bees inside the hive</li>
 
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<li>Take a frame out the hive and remove bees with big brush and smoker</li>
 
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<li>Close bee hive</li>
 
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<li>In the lab, put the frame on a table against the wall</li>
 
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<li>With tweezer drill hole into one beehive cell</li>
 
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<li>Remove larvae and look for varroas on larvae and on beehive cell</li>
 
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<li>If there are varroas, take them with a small brush and put them on Petri dishes</li>
 
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<li>Make sure there are two or three larvae on Petri dishes in order to allow survival of varroas</li>
 
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<li>Start again step 7 to 9 until you have enough varroas</li>
 
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</ol>
 
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</div>
 
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</div>
 
  
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/* Latest
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<div class="subtitle">  
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<h3>Standardization of varroas and sampling</h3>
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<div class="group center">
 
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<p align="justify" style="font-size:15px;">When we take varroas directly from frame, as it is described in
 
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protocol “Sampling Varroas”, we have varroas in different phases. In order to have varroas
 
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in the same phase it is necessary to add one step and it is important for reproducibility of
 
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experiments. With this method we place varroas on adult bees so all varroas will be in phoretic phase.</p>
 
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</div>
 
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<div class="group" style="padding-top:10px">
 
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<div class="one_quarter first">
 
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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>
 
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</div>
 
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<ul align="justify" style="font-size:15px;">
 
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<li>Bees in box with aeration and glucose </li>
 
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<li>Varroas from protocol “Sampling varroas”</li>
 
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<li>Gas cylinder of CO<SUB>2</SUB></li>
 
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<li>Small brush </li>
 
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<li>Tweezers</li>
 
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<li>Petri dishes <br> Ø x h = 35 x 15 mm</li>
 
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</div>
 
  
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/* Clients
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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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.clients ul li:first-child, .clients ul li:nth-child(5n+1){clear:left;}
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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 *{width:100%; opacity:.5;}
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<li>Take the box out of incubator</li>
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.clients ul li *:hover{opacity:1;}
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<li>Add CO<SUB>2</SUB> from gas cylinder into the box until all bees fall down</li>
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−
<li>Open the box, take a bee with tweezer and look for varroas</li>
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−
<li>When you find a varroa take him with small brush and replace bee in the box</li>
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−
<li>Start again step 5 and 6 until you have enough varroas</li>
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−
</ol>
+
−
</div>
+
−
</div>
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−
<div class="group">
 
−
<div class="subtitle" >  
 
−
<h3>Attraction test on varroas</h3>
 
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</div>
 
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</div>
 
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<div class="group center">
 
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<p align="justify" style="font-size:15px;">In order to test the attraction effect of butyric acid
 
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on varroas an Y test was used, as it is showed with diagram below. Where we place varroas for the
 
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test we choose glass pipe because on plastic they could load themselves with electrostatics and die.
 
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For butyric acid concentration we choose 4% (V/V) because this is the concentration used in the patent we see, see “Attribution” part. </p>
 
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</div>
 
  
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<h3 style="font-size:20px;">Materials</h3>
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<ul align="justify" style="font-size:15px;">
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<li>Pump wich expels air</li>
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<li>15 mL Flacon tube</li>
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<li>Plastic pipe <br> Ø = 10mm</li>
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−
<li>Glass T pipe <br> Ø = 10mm, made by a glassworker</li>
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<li>Plastic separator</li>
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<li>Carded cotton</li>
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<li>Absorbent cotton</li>
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<li>5mL 4% (V/V) Butyric acid</li>
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<li>5mL Water</li>
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<li>Standardized varroas <br></li>
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<h3 style="font-size:20px;">Methods</h3>
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</div>
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<ol align="justify" style="font-size:15px;">
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<li>Put a cotton on Petri dish and add 400µL of one acid formic solution</li>
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<li>Place three varroas on this Petri dish and close it</li>
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−
<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>
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−
<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>
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<li>Two tests can be made in the same time thanks to the plastic separator</li>
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</ol>
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</div>
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</div>
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 +
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<div class="subtitle" >  
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.container .sidebar{}
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<h3>Mortality test on varroas</h3>
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</div>
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</div>
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<div class="group center">
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<p align="justify" style="font-size:15px;">To test toxicity of formic acid
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on varroas we had to choose which concentrations we use. For that we based
+
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our thoughts on present treatments. When beekeepers use formic acid for long
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treatment they place a diffuser at the top of the hive and formic acid concentration
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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>.
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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.
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</p>
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</div>
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 +
.sidebar .sdb_holder{margin-bottom:50px;}
 +
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<p style="font-size:15px;">
 
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$$ P\cdot V = n\cdot R\cdot T, \textrm{ideal gaz law} $$
 
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<!-- P. V = n.R.T, ideal gaz law-->
 
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$$ P_A = C_A\cdot R\cdot T = 7,826\cdot10^{-3}\times8,314\times293=19,964 Pa $$
 
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</p>
 
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</div>
 
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<ul style="font-size:15px;">
 
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<li>P<SUB>A</SUB>: partial pressure of A in Pa</li>
 
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<li>C<SUB>A</SUB>: Concentration of A in air in mol.m<SUP>-3</SUP></li>
 
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<li>R: perfect gaz constant = 8,314 J.mol<SUP>-1</SUP>.K<SUP>-1</SUP></li>
 
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<li>T: temperature in <SUP>°</SUP>K </li>
 
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</ul>
 
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<br>
 
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<div class="group center" style="padding-top:10px;">
 
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<p style="font-size:15px;">
 
−
<!-- P_A = C_A.R.T = 7,826.10^3 x 8,314 x 293 = 19,964 Pa -->
 
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$$ P_A = H_A\cdot C_{A,eq}, \textrm{Henry's law} $$
 
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<!-- P_A = H_A . C_{A,eq}, Henry's law -->
 
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$$ C_{A,eq} = \frac{19,964}{0,019} = 1,019mol.L^{-1}$$
 
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</p>
 
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</div>
 
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<ul style="font-size:15px;">
 
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<li>C<SUB>A,eq</SUB>: equivalent concentration in liquid in mol.L<SUP>-1</SUP></li>
 
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<li>H<SUB>A</SUB>: Henry's constant = 0,019 Pa.m<SUP>3</SUP>mol<SUP>-1</SUP></li>
 
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</ul>
 
  
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<div class="one_quarter first">
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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>
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</div>
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<ul align="justify" style="font-size:15px;">
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<li>Petri dishes <br> Ø x h = 35 x 15 mm</li>
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<li>Varroas form “Sampling varroas”</li>
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<li>Cotton</li>
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<li>Acid formic solutions : </li>
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<ul align="justify" style="font-size:15px;">
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<li>2 mol.L<SUP>-1</SUP></li>
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<li>10 mmol.L<SUP>-1</SUP></li>
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<li>1 mmol.L<SUP>-1</SUP></li>
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<li>500 µmol.L<SUP>-1</SUP></li>
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<li>50 µmol.L<SUP>-1</SUP></li>
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</ul>
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<li>Water</li>
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</div>
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<h3 style="font-size:20px;">Methods</h3>
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</div>
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<ol align="justify" style="font-size:15px;">
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<li>Put a cotton on Petri dish and add 400µL of one acid formic solution</li>
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<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>
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−
<li>Each 30 minutes check if varroas are alive. To do that : </li>
+
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<ol align="justify" style="font-size:15px;">
+
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<li value="5">Tap on Petri dish and see if varroa move. If yes varroa is alive, if not see below</li>
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<li>Look at binocular magnifier if varroas move their feets. If yes varroa is alive, if not varroa is dead <br>
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Remark: Varroa stop moving approximately one or two hours before it die </li>
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</li>
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</ol>
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#footer .smallfont{font-size:.8em; margin:0;}
  
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#footer input, #footer button{border:none; border-radius:5px;}
 +
#footer input{display:block; width:100%; padding:8px;}
 +
#footer button{padding:6px 12px; font-weight:bold; text-transform:uppercase; cursor:pointer;}
  
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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 -->
 
  
  
−
<div class="subtitle">
+
/* Copyright
−
    <h3> Preparation of Competent Cells </h3>  <!-- COMPETENT CELLS -->
+
---------------------------------------------------------------------------------------------------------------*/
−
    </div>
+
#copyright{padding:30px 0;}
−
<div class="group">
+
#copyright p{margin:0; padding:0;}
−
<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">
 
  
−
<ul align="justify" style="font-size:15px;">
+
/* Transition Fade
−
<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>
+
--------------------------------------------------------------------------------------------------------------- */
−
<li>2. Resuspend one colony per culture tube in 5 mL of LB medium with antibiotic</li>
+
.row0, .faico a, #mainav *, .shout-content, .overlay ::before, .overlay ::after, .boxedicon a, .clients ul 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> 
 
  
−
<div class="group">
+
/* Navigation
−
<ul align="justify" style="font-size:15px;">
+
---------------------------------------------------------------------------------------------------------------*/
−
<li>1. Prepare a 1% or 2% electrophoresis agarose gel with 0.5x TAE buffer </li>
+
nav ul, nav ol{margin:0; padding:0; list-style:none;}
−
<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> 
+
  
−
<div class="group">
+
/* Top Navigation */
−
<ul align="justify" style="font-size:15px;">
+
#mainav{margin-bottom:-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{text-transform:uppercase;margin-top:-1px;}
−
<li>2. Plate the solution on selective medium overnight at 37°C</li>
+
#mainav ul ul{z-index:9999; position:absolute; width:160px; text-align:left; text-transform:none;}
−
</ul>
+
#mainav ul ul ul{left:150px; top:0;}
−
</div>
+
#mainav li{display:inline-block; position:relative; margin:0 10px 0 0; padding:0;}
−
 
+
#mainav li:last-child{margin-right:0;}
−
  </main>
+
#mainav li li{width:100%; margin:0;}
−
</div>
+
#mainav li a{display:block; padding:25px 10px;}
−
<!-- ################################################################################################ -->
+
#mainav li li a{border:solid; border-width:0 0 1px 0;font-size:12px;}
−
<!-- ################################################################################################ -->
+
#mainav .drop{padding-left:15px; margin-top:20px;}
−
<!-- ################################################################################################ -->
+
#mainav li li a, #mainav li li .drop{display:block; margin:0px; padding:10px 15px;}
 +
#mainav .drop::after, #mainav li li .drop::after{content:"";}
 +
#mainav .drop::after{top:30px; left:5px;}
 +
#mainav li li .drop::after{top:15px; left:5px;}
 +
#mainav ul ul{visibility:hidden; opacity:0;}
 +
#mainav ul li:hover > ul{visibility:visible; opacity:1;}
  
−
<!-- ################################################################################################ -->
+
/* Breadcrumb */
−
<!-- ################################################################################################ -->
+
#breadcrumb{display:block; padding:15px 20px; border-radius:5px 5px 0 0;}
−
<!-- ########################### SPONSORS ###################################################################### -->
+
#breadcrumb ul{margin:0; padding:0; list-style:none;}
−
<div class="wrapper row4">
+
#breadcrumb li{display:inline-block; margin:0 6px 0 0; padding:0;}
 +
#breadcrumb li a{display:block; position:relative; margin:0; padding:0 12px 0 0; font-size:13px;color:rgba(29, 5, 79, 1);}
 +
#breadcrumb li a::after{top:5px; right:0; content:"";}
 +
#breadcrumb li:last-child a{margin:0; padding:0;}
 +
#breadcrumb li:last-child a::after{display:none;}
  
−
<div class="container clear" style="padding-top:30px;">
+
/* Sidebar Navigation */
−
<center><p class="maintitle">  
+
.sidebar nav{display:block; width:100%;}
−
References
+
.sidebar nav li{margin:0 0 3px 0; padding:0;}
−
</p></center>
+
.sidebar nav a{display:block; position:relative; margin:0; padding:5px 10px 5px 15px; text-decoration:none; border:solid; border-width:0 0 1px 0;}
−
<br>
+
.sidebar nav a::after{top:9px; left:5px; content:"";}
−
<div class="clear">
+
.sidebar nav ul ul a{padding-left:35px;}
 +
.sidebar nav ul ul a::after{left:25px;}
 +
.sidebar nav ul ul ul a{padding-left:55px;}
 +
.sidebar nav ul ul ul a::after{left:45px;}
  
−
</div>
+
/* Pagination */
−
<br>
+
.pagination{display:block; width:100%; text-align:center; clear:both;}
−
</div>
+
.pagination li{display:inline-block; margin:0 3px 0 0;}
 +
.pagination li:last-child{margin-right:0;}
 +
.pagination a, .pagination strong{display:block; padding:8px 11px; border:1px solid; font-weight:normal;}
  
−
</div>
 
−
<!-- ################################################################################################ -->
 
−
<!-- ################################################################################################ -->
 
−
<!-- #######################        FOOTER        ######################################################################### -->
 
  
−
</html>{{Toulouse/footer}}<html>
+
/* Forms
 +
---------------------------------------------------------------------------------------------------------------*/
 +
form, fieldset, legend{margin:0; padding:0; border:none;}
 +
legend{display:none;}
 +
label, input, textarea, select{display:block; resize:none; font-size:inherit; box-sizing:border-box;}
 +
label{margin-bottom:5px;}
 +
 
 +
/* Comment Area */
 +
#comments form{display:block; width:100%;}
 +
#comments input, #comments textarea{width:100%; padding:10px; border:1px solid;}
 +
#comments textarea{overflow:auto;}
 +
#comments div{margin-bottom:15px;}
 +
#comments input[type="submit"], #comments input[type="reset"]{display:inline-block; width:auto; min-width:150px; margin:0; padding:8px 5px; cursor:pointer;}
 +
 
 +
 
 +
/* Tables
 +
---------------------------------------------------------------------------------------------------------------*/
 +
table.df th td{border:1px solid; border-collapse:collapse; vertical-align:top; box-sizing:border-box;}
 +
table.df th{table-layout:auto;}
 +
table.df{width:100%; margin-bottom:15px;}
 +
table.df th td{padding:10px 15px;}
 +
table.df td{border-width:0 1px;}
 +
 
 +
 
 +
/* Gallery
 +
---------------------------------------------------------------------------------------------------------------*/
 +
#gallery{display:block; width:100%; margin-bottom:50px;}
 +
#gallery figure figcaption{display:block; width:100%; clear:both;}
 +
#gallery li{margin-bottom:30px;}
 +
 
 +
.overlay ul li a{display:block; position:relative;}
 +
.overlay ul li a::before{display:block; position:absolute; top:0; left:0; width:100%; height:100%; content:"";}
 +
.overlay ul li a::after{display:block; position:absolute; top:50%; left:50%; width:40px; height:40px; line-height:40px; margin:-18px 0 0 -18px; padding:0; border-radius:50%; font-family:"FontAwesome"; content:""; font-size:18px; text-align:center;}
 +
.overlay ul li a::before, .overlay ul li a::after{opacity:0;}
 +
.overlay ul li a:hover::before, .overlay ul li a:hover::after{opacity:1;}
 +
 
 +
 
 +
/* Font Awesome Social Icons
 +
---------------------------------------------------------------------------------------------------------------*/
 +
.faico{margin:40px; padding:0; list-style:none;}
 +
.faico li{display:inline-block; margin:0 2px 0 0; padding:0; line-height:normal;}
 +
.faico li:last-child{margin-right:0;}
 +
.faico a{display:inline-block; width:30px; height:30px; line-height:30px; border-radius:5px; font-size:16px; text-align:center;}
 +
 
 +
.faico a{color:inherit; }
 +
.faico a:hover{color:#FFFFFF;}
 +
 
 +
.faicon-facebook:hover{background-color:#3B5998;}
 +
.faicon-google-plus:hover{background-color:#DB4A39;}
 +
.faicon-instagram:hover{background-color:#3F729B;}
 +
.faicon-linkedin:hover{background-color:#0E76A8;}
 +
.faicon-tumblr:hover{background-color:#34526F;}
 +
.faicon-twitter:hover{background-color:#00ACEE;}
 +
 
 +
 
 +
/*-------------------------------------------------------------------------------------------------------------*/
 +
/*-------------------------------------------------------------------------------------------------------------*/
 +
/*-------------------------------------------------------------------------------------------------------------*/
 +
/*-------------------------------------------------------------------------------------------------------------*/
 +
/*-------------------------------------------------------------------------------------------------------------*/
 +
 
 +
 
 +
/* Colours
 +
---------------------------------------------------------------------------------------------------------------*/
 +
body{color:black; background-color:#817E8B;}
 +
 
 +
hr, .borderedbox{border-color:#EDEDED;}
 +
label span{color:#FF0000; background-color:inherit;}
 +
.btn{color:#FFFFFF;}
 +
.font_xxl:after{color:inherit; background-color:rgba(9, 181, 31, 0.32);}
 +
 
 +
 
 +
/* Rows */
 +
/* Structure des rows, le row0 est la barre de nav */
 +
.row0{color:#101132; background-color:rgba(245, 245, 245, 1); border-color:rgba(29, 5, 79, 0.81); max-height:630px}
 +
.row0:hover{background-color:rgba(245, 245, 245, 1);}
 +
.row1, .row1 a{color:#FFFFFF; background-color:#000000;}
 +
.row2, .row2 a{color:#FFFFFF; background-color:#000000;}
 +
.row3{color:#888888; background-color:white;}
 +
.row4, .row4 a{color:#B4B4B4; background-color:#EDEDED;}
 +
.row5{color:white; background-color:#817E8B;}
 +
.row6, .row6 a{color:#B4B4B4; background-color:#817E8B;}
 +
.row6{border-color:#3C3C3C;}
 +
 
 +
 
 +
/* Header */
 +
#header #logo h1 a{color:inherit;}
 +
 
 +
 
 +
/* Shout */
 +
.shout .shout-content{color:inherit; background-color:rgba(247, 244, 255, 0.48);max-width:800px;max-height:300px;}
 +
.shout .shout-content:hover{background-color:rgba(247, 244, 255, 0.48);}
 +
 
 +
 
 +
/* Latest Work */
 +
.latest ul, .latest ul li{border-color:#EDEDED;}
 +
 
 +
 
 +
/* Content */
 +
.boxedicon a{color:#56AED4; }
 +
.boxedicon a:hover{color:#FFFFFF;}
 +
 
 +
 
 +
/* Footer */
 +
/* Couleur de la barre du footer */
 +
#footer .shout .shout-content, #footer .shout .shout-content:hover{color:rgba(8, 44, 47, 0.82); background-color:#EDEDED; border-bottom: 7px solid rgba(46, 29, 89, 0.88);}
 +
#footer .title{color:#FFFFFF; border-color:#3C3C3C;} /* Couleur des textes */
 +
#footer input{color:#FFFFFF; background-color:#3C3C3C;}
 +
#footer button{color:#FFFFFF;}
 +
e-block;}
 +
 
 +
/*Back to Top*/
 +
 
 +
 
 +
/* Navigation */
 +
 
 +
#mainav li a{color:inherit;} /* Couleur des textes de la barre nav */
 +
#mainav.active a, #mainav a:hover, #mainav li:hover > a{; background-color:inherit;} /* Couleur de fond lors d'un mouseover du texte */
 +
#mainav li li a, #mainav .active li a{color:black; background-color:rgb(144, 168, 62); border-color:rgb(144, 168, 62);} /* Gestion de la couleur des onglets */
 +
#mainav li li:hover > a, #mainav .active .active > a{color:#FFFFFF; background-color:rgba(29, 5, 79, 0.81);border-color:rgba(29, 5, 79, 0.81);font-size:12px;}/* Gestion couleur des onglets lors d'un mouseover */
 +
#mainav li a.set{color:#90A83E;font-size:15px;} /*classe active qui s'appelle pas active parceque active ?a marchait pas */
 +
/* J'ai toujours pas trop compris */
 +
#breadcrumb{color:inherit; background-color:rgba(255, 255, 255, 0.7);}
 +
#breadcrumb a{background-color:inherit;}
 +
 
 +
.container .sidebar nav a{color:inherit; border-color:#EDEDED;}
 +
.container .sidebar nav a:hover{color:#56AED4;}
 +
 
 +
.pagination a, .pagination strong{border-color:#EDEDED;}
 +
.pagination .current{color:#B9D94A;}
 +
 
 +
/*ALL TABLE ATTRIBUTES*/
 +
/* Tables + Comments
 +
df for default style table*/
 +
table.df th td, #comments .avatar, #comments input, #comments textarea{border-color:#EDEDED;}
 +
table.df th{color:#FFFFFF; background-color:rgba(29, 5, 79, 1);font-size:15px;}
 +
table.df tr, #comments li, #comments input[type="submit"], #comments input[type="reset"]{color:inherit; background-color:#FBFBFB;}
 +
table.df tr:nth-child(even), #comments li:nth-child(even){color:inherit; background-color:#F7F7F7;}
 +
table.df a, #comments a{background-color:inherit;}
 +
 
 +
 
 +
/* Gallery */
 +
.overlay ul li a::before{background-color:rgba(236, 255, 226, 0.6);}
 +
.overlay ul li a::after{color:#FFFFFF; }
 +
 
 +
/* */
 +
a:link {color: rgba(29, 5, 79, 0.81);}
 +
a:visited {color: rgba(29, 5, 79, 0.81);}
 +
a:hover {color:rgba(22, 19, 29, 0.95);}
 +
a:active {color:rgba(29, 5, 79, 0.81);}
 +
img
 +
{
 +
    filter: type(value);
 +
}
 +
img
 +
{
 +
    filter: type(value);
 +
    -webkit-filter: type(value);
 +
    -moz-filter: type(value);
 +
    -ms-filter: type(value);
 +
    -o-filter: type(value);
 +
}
 +
.grayscale img:hover
 +
{
 +
        filter: grayscale(0);
 +
        -webkit-filter: grayscale(0);
 +
        -moz-filter: grayscale(0);
 +
        -o-filter: grayscale(0);
 +
        -ms-filter: grayscale(0);
 +
}
 +
.grayscale img
 +
{
 +
        filter: grayscale(1);
 +
        -webkit-filter: grayscale(1);
 +
        -moz-filter: grayscale(1);
 +
        -o-filter: grayscale(1);
 +
        -ms-filter: grayscale(1);
 +
}
 +
 
 +
.group {padding-top:10px;}
 +
 
 +
 
 +
.subtitle{
 +
right: 5px;
 +
padding-top:30px;
 +
margin-top:30px;
 +
}
 +
 
 +
.title h3{
 +
color:rgba(29, 5, 79, 1);
 +
font-size:25px;
 +
}
 +
 
 +
.subtitle h3{
 +
color:#90A83E;
 +
font-size:25px;
 +
}
 +
 
 +
 
 +
.title{
 +
font-size:25px;
 +
color:rgba(29, 5, 79, 0.81);
 +
}
 +
 
 +
.maintitle{
 +
 
 +
font-size:30px;
 +
color:rgba(29, 5, 79, 0.81);
 +
}
 +
 
 +
p.legend{
 +
font-size:78%;
 +
margin-bottom:20px;
 +
padding-bottom:10px;
 +
}
 +
 
 +
 
 +
table.team{
 +
text-align:center;
 +
background-color:rgb(0,0,0,0); /*transparent*/
 +
 
 +
}
 +
 
 +
table.team td {
 +
padding-top: 15px;
 +
padding-bottom: 15px;
 +
width:472px;
 +
height:327px;
 +
}
 +
 
 +
 
 +
 
 +
table.team td img{
 +
width:472px;
 +
height:327px;
 +
}
 +
 
 +
 
 +
 
 +
table.result{
 +
  display: block;
 +
  font-family: sans-serif;
 +
  -webkit-font-smoothing: antialiased;
 +
  font-size: 115%;
 +
  overflow: auto;
 +
  width: auto;
 +
  }
 +
 +
table.result th {
 +
    background-color: rgb(144, 168, 62);
 +
    color: white;
 +
    font-weight: normal;
 +
    padding: 20px 30px;
 +
    text-align: center;
 +
  }
 +
 
 +
table.result  td {
 +
    background-color: rgb(238, 238, 238);
 +
    color: rgb(111, 111, 111);
 +
    padding: 20px 30px;
 +
  }
 +
 
 +
 
  
 +
.anchor {
 +
    height: 30px;
 +
    position: fixed;
 +
    bottom: 30px;
 +
    right: 30px;
 +
    z-index: 1000;
 +
    cursor: pointer;
 +
    -webkit-transition: all ease 0.4s;
 +
    -moz-transition: all ease 0.4s;
 +
    -ms-transition: all ease 0.4s;
 +
    -o-transition: all ease 0.4s;
 +
    transition: all ease 0.4s;
 +
}
 +
/* SLIDESHOW */
  
 +
    .contener_slideshow
 +
    {
 +
      width:800px;
 +
      height:600px;
 +
      overflow: hidden;
 +
      position: relative;
 +
      background-image: url(YOUR-URL-IMAGE.png);
 +
    }
 +
    .slid_1, .slid_2, .slid_3
 +
    {
 +
      position: absolute;
 +
      width:800px;
 +
      height:600px;
 +
    }
 +
    .slid_1{left: 0;}
 +
    .slid_2{left: 800px;}
 +
    .slid_3{left: 1600px;}
 +
    .contener_slide
 +
    {
 +
      width: 2400px;
 +
      height: 600px;
 +
      left:0px;
 +
      position: absolute;
 +
      -webkit-animation-duration: 10s;
 +
      -webkit-animation-iteration-count:infinite;
 +
      -webkit-animation-name: anim_slide;
 +
      -moz-animation-duration: 10s;
 +
      -moz-animation-iteration-count:infinite;
 +
      -moz-animation-name: anim_slide;
 +
      -ms-animation-duration: 10s;
 +
      -ms-animation-iteration-count:infinite;
 +
      -ms-animation-name: anim_slide;
 +
      animation-duration: 10s;
 +
      animation-iteration-count:infinite;
 +
      animation-name: anim_slide;
 +
    }
 +
    @-webkit-keyframes anim_slide
 +
    {
 +
      0% {left:0px;}
 +
      22% {left:0px;}
 +
      33% {left:-800px;}
 +
      45% {left:-800px;}
 +
      66% {left:-1600px;}
 +
      90% {left:-1600px;}
 +
    }
 +
    @-moz-keyframes anim_slide
 +
    {
 +
      0% {left:0px;}
 +
      22% {left:0px;}
 +
      33% {left:-800px;}
 +
      45% {left:-800px;}
 +
      66% {left:-1600px;}
 +
      90% {left:-1600px;}
 +
    }
 +
    @-ms-keyframes anim_slide
 +
    {
 +
      0% {left:0px;}
 +
      22% {left:0px;}
 +
      33% {left:-800px;}
 +
      45% {left:-800px;}
 +
      66% {left:-1600px;}
 +
      90% {left:-1600px;}
 +
    }
 +
    @keyframes anim_slide
 +
    {
 +
      0% {left:0px;}
 +
      22% {left:0px;}
 +
      33% {left:-800px;}
 +
      45% {left:-800px;}
 +
      66% {left:-1600px;}
 +
      90% {left:-1600px;}
 +
    }
  
−
</body>
+
</style>
 
</html>
 
</html>

Revision as of 14:30, 2 September 2015