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| − | <h1><center>Experiments</center></h1>
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| − | <h3><b><center>Protocols!</center></b></h3>
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| − | <h3><b>STX in Buffer HEPES</b></h3>
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| − | <p>
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| − | A master mix for each aptazyme sample was prepared following these instructions:
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| − | <li>110 uL of HEPES Buffer 10x</li>
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| − | <li>11 uL DNA</li>
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| − | <li>698.5 uL of nuclease free water</li>
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| − | </p>
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| − | <p>At the end of this procedure we obtained 7 different master mixes for each one of our samples (this includes de control without DNA).
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| − | After this, each of these master mixes was heated in a dry bath for 5 minutes at 90ºc and after that they were left cooling down at room temperature for 25 minutes.
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| − | In the meantime every reactive was prepared in a way that the final concentrations of each reactive in the final solution 8the one placed in every plate well) were as following:</p>
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| − | <li>-Hydrogen peroxyde 2mM</li>
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| − | <li>-ABTS 5 mM</li>
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| − | <li>-Hemin 0,375 uM</li>
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| − | <p>
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| − | When the master mixes were ready, we poured in every plate well the following volumes:
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| − | </p>
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| − | <li>-10 uL Hidrogen peroxyde</li>
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| − | <li>-5 uL ABTS</li>
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| − | <li>-7,5 uL Hemin</li>
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| − | <li>- X uL STX (figure below as refence)</li>
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| − | <li>-74, ul from the corresponding master mix</li>
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| − |
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| − | <p>The last reactive used was the Hydrogen peroxide because its function as the initiator and it was poured after 2 hours in order to let the Aptazyme interact with the toxin in addition only in the wells needed the volumes were adjusted with nuclease-free water to reach the 100 uL for the reaction!
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| − | The Hydrogen peroxide was poured one row at a time, to prevent possible errors associated to the reaction rate constant, as it only takes about 3 minutes to be complete.</p>
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| − | <p>
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| − | To measure the absorbance we used a Infinite 200 PRO NanoQuant Plate Reader from Tecan Trading AG. We measured the changes in the absorbance levels for 20 cycles every 30 seconds.
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| − | </p>
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| − | <center><img src="https://static.igem.org/mediawiki/2017/6/62/Protocolo_25-10.jpg" alt="figure 1. Experiment design Buffer HEPES">
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| − | </img></center>
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| − | <div class="clear">
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| − | </div>
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| − |
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| − | <h3> in Buffer HEPES and Buffer PBST</h3>
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| − |
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| − | <p>Two sets of master mixes were made for each sample analyzed; these master mixes were made as followed:</p>
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| − | <li>-90 uL of Buffer</li>
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| − | <li>-9 uL of DNA</li>
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| − | <li>-130.5 uL nuclease free water</li>
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| − |
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| − | <p>Once the 2 sets of master mixes were ready, they were heated at 90ºc for 5 minutes and cooled down at room temperature for 25 minutes, in the meantime each reactive was prepared the same way as seen in the previous protocol for bufffer HEPES
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| − | </p>
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| − | <p>As the concentrations of STX were different for each set of palte wells, we made 3 different preparations:</p>
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| − | <div class="column half_size">
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| − | <p><b> B. STX</b></p>
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| − | <li>-1 uL DNA</li>
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| − | <li>-7.5 uL Hemin</li>
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| − | <li>-10 uL Hidrogen peroxyde</li>
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| − | <li>-5 uL ABTS</li>
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| − | <li>-10 uL Buffer</li>
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| − | <li>-69.5 uL nuclease free water</li>
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| − | </div>
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| − | <div class="column half_size">
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| − | <p><b> 0.33 uM STX</b></p>
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| − | <li>-1 uL DNA</li>
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| − | <li>-7.5 uL Hemin</li>
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| − | <li>-10 uL Hidrogen peroxyde</li>
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| − | <li>-5 uL ABTS</li>
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| − | <li>-10 uL Buffer</li>
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| − | <li>-27.5 uL nuclease free water</li>
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| − | <li>39 uL STX</li>
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| − | </div>
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| − |
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| − | <p><b> 0.45 uM STX</b></p>
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| − | <li>-1 uL DNA</li>
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| − | <li>-7.5 uL Hemin</li>
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| − | <li>-10 uL Hidrogen peroxyde</li>
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| − | <li>-5 uL ABTS</li>
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| − | <li>-10 uL Buffer</li>
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| − | <li>-14.5 uL nuclease free water</li>
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| − | <li>-52 uL STX</li>
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| − |
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| − | <p>The absorbance was measured with an Infinite 200 PRO NanoQuant Plate Reader from Tecan Trading AG in a period of 570 seconds. Each measure was made every 30 seconds.
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| − | </p>
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| − | <center><img src="https://static.igem.org/mediawiki/2017/d/d3/HEPESyPBST-STX.jpg" alt="Figure 2. plate diagram used for this protocol"></img></center>
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| − |
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| − | <p><center><b> Note: The circles marked with a cross were not prepared, and the plates with AMP 5mM were solely made with buffer HEPES</b></center></p>
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| − |
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| − | <h3>Thermocycler configuration</h3>
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| − | <p>
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| − | The configuration used for the Thermocycler (only when needed) was the following:
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| − | <li>-90ºC for 5 minutes</li>
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| − | <li>-76ºC for 5 minutes</li>
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| − | <li>-62ºC for 5 minutes</li>
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| − | <li>-48ºC for 5 minutes</li>
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| − | <li>-32ºC for 5 minutes</li>
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