Line 268: | Line 268: | ||
<p class="lead nomargin"><spam class="red">CAUTION</spam>: As the thiolated ends are considerably unstable, they are shipped as they oxidized form. To treat your electrodes with this aptamers you need to reduce them with DTT or TCEP. You could find a complete protocol of this process <a href="https://sfvideo.blob.core.windows.net/sitefinity/docs/default-source/protocol/reduction-protocol-for-thiol-modified-oligonucleotides.pdf">here.</a></p> | <p class="lead nomargin"><spam class="red">CAUTION</spam>: As the thiolated ends are considerably unstable, they are shipped as they oxidized form. To treat your electrodes with this aptamers you need to reduce them with DTT or TCEP. You could find a complete protocol of this process <a href="https://sfvideo.blob.core.windows.net/sitefinity/docs/default-source/protocol/reduction-protocol-for-thiol-modified-oligonucleotides.pdf">here.</a></p> | ||
− | <h4 class="tittlelist"> | + | <h4 class="tittlelist">Aptamer Bounding</h4> |
− | <li class="nomargin"> <p class="lead">[Optional] Depending on your electrodes, it needs to be pre-treated to ensure the correct aptamer binding. For this purpose pipette 50 uL of H2SO4 0.5M until the electrode are covered and perform 10 cyclic voltammograms from 0V to 1.25V at 100 mV/s of scan rate | + | <li class="nomargin"> <p class="lead">[Optional] Depending on your electrodes, it needs to be pre-treated to ensure the correct aptamer binding. For this purpose pipette 50 uL of H2SO4 0.5M until the electrode are covered and perform 10 cyclic voltammograms from 0V to 1.25V at 100 mV/s of scan rate.</p></li> |
<p class="lead nomargin"><spam class="purple">ADVICE</spam>: TWith Dropsens electrodes there is no need to perform this step.</p> | <p class="lead nomargin"><spam class="purple">ADVICE</spam>: TWith Dropsens electrodes there is no need to perform this step.</p> | ||
<li class="nomargin"><p class="lead">Wash the electrodes three times with deionized water and let them dry under an extraction hood air flow.</p></li> | <li class="nomargin"><p class="lead">Wash the electrodes three times with deionized water and let them dry under an extraction hood air flow.</p></li> | ||
Line 281: | Line 281: | ||
<p class="lead nomargin"><spam class="red">CAUTION</spam>: When incubating the different solutions and buffers with the electrode, do NOT let the solution evaporate. Be sure of making the step in a humidified chamber.</p> | <p class="lead nomargin"><spam class="red">CAUTION</spam>: When incubating the different solutions and buffers with the electrode, do NOT let the solution evaporate. Be sure of making the step in a humidified chamber.</p> | ||
+ | <h4 class="tittlelist">Electrodes testing with cyclic voltammetry</h4> | ||
+ | <li class="nomargin"> <p class="lead">First of all you must calibrate the ideal concentration of the electrode donor solution. For this purpose ferricyanide redox couple (K3Fe(CN)6and K4Fe(CN)6) was used above a raw electrode without aptamer. After our experiments the optimal concentration was found to be 5mM of each chemical in a 0.1 KCl solution.</p></li> | ||
+ | <p class="lead nomargin"><spam class="purple">ADVICE</spam>: In our experience, this concentration could be different depending on things like the quality of your reactives or your electrodes. We encourage you to adjust this value experimentally making some dilutions (0.5X, 2X…).</p> | ||
+ | <img alt="Image1" src="https://static.igem.org/mediawiki/2018/a/a3/T--Madrid-OLM--Experiments--Protocols_--Aptamers--Maquina.jpg" style="width:30%;"/> | ||
+ | <li class="nomargin"><p class="lead">Cover the electrode with a ferricyanide droplet and connect it to the potentiostat.</p></li> | ||
+ | <li class="nomargin"><p class="lead">Run a preliminar cyclic voltammetry test with a stardart parameters. The ones that have fits better with our hardware <a href="https://sfvideo.blob.core.windows.net/sitefinity/docs/default-source/protocol/reduction-protocol-for-thiol-modified-oligonucleotides.pdf">(Rodeostat)</a> was one cycle from -0.3V to 0.3V, with a current limit of 1000 uA, a sample rate of 100 Hz and a scan rate of 0.05 mV/s.</p></li> | ||
+ | <li class="nomargin"><p class="lead">After the test have finished, adjust the parameters (voltage range and current limit) to fit the complete curve in your range. A typical Cyclic Voltammetry curve may have a shape similar to a duck.</p></li> | ||
+ | <img alt="Image1" src="https://static.igem.org/mediawiki/2018/1/16/T--Madrid-OLM--Experiments--Protocols_--Aptamers--Duck1.jpg" style="width:80%;"/> | ||
+ | |||
+ | <p class="lead"><spam class="green">PAUSE POINT</spam>: Let the electrodes incubating overnight</p> | ||
+ | <li class="nomargin"><p class="lead">Wash the electrodes three times with deionized water and let them dry under an extraction hood air flow.</p></li> | ||
+ | <li class="nomargin"><p class="lead">To remove the excess of DNA, treat the electrodes with 10 uL of β-Mercaptoethanol for 50 minutes in a humidity chamber. </p></li> | ||
+ | <li class="nomargin"><p class="lead">Wash the electrodes three times with deionized water and let them dry under an extraction hood air flow.</p></li> | ||
+ | <p class="lead nomargin"><spam class="red">CAUTION</spam>: When incubating the different solutions and buffers with the electrode, do NOT let the solution evaporate. Be sure of making the step in a humidified chamber.</p> | ||
</ol> | </ol> | ||
</div> | </div> |
Revision as of 09:21, 12 October 2018
Aptamer's Protocols
Texto de explicacion/ resumen de la pagina.
Aptamer Characterization
Elona
Aptamer electrode
Sinthesis of the electrode
SELEX
Bill Of Materials: You could see a complete BoM here (upload the bill).
Amount of time: 2 day
Total costs: calcular con BoM
-
We choose Dropsens as our provider, because they are one of the standards in the field, and they are relatively near to our laboratory.
The material of the working electrode was choose as carbon, modified to include gold nanoparticles. The carbon have better electrochemical window than gold or silver (check this post for more information) and gold are the ideal substrate to join DNA (It only have to be thiolated).
-
Between the DNA and its thiolation in its 5’, we have include a 6 carbon chain after the thiol modification and 15 thymes before the aptamer sequence. The purpose of this modifications was to separate the aptamer from the electrode surface aiming to ensure enough conformational flexibility of the molecule.
We have order the aptamers to Integrated DNA Technologies as they are one of the competition sponsors.
-
[Optional] Depending on your electrodes, it needs to be pre-treated to ensure the correct aptamer binding. For this purpose pipette 50 uL of H2SO4 0.5M until the electrode are covered and perform 10 cyclic voltammograms from 0V to 1.25V at 100 mV/s of scan rate.
Wash the electrodes three times with deionized water and let them dry under an extraction hood air flow.
Follow the protocol to structure the aptamers in their individual binding buffers. If you have follow our SELEX protocol, check the buffers and their own structuration steps in this protocol. Make sure that you have enough concentration for the next step.
Drop 10 uL of the 5uM solution of aptamer in its own Binding Buffer (if you have selected the aptamer with our protocol check the SELEX protocol) above the working electrode.
Incubate overnight in an humidity chamber.Incubate overnight in an humidity chamber.
Wash the electrodes three times with deionized water and let them dry under an extraction hood air flow.
To remove the excess of DNA, treat the electrodes with 10 uL of β-Mercaptoethanol for 50 minutes in a humidity chamber.
Wash the electrodes three times with deionized water and let them dry under an extraction hood air flow.
-
First of all you must calibrate the ideal concentration of the electrode donor solution. For this purpose ferricyanide redox couple (K3Fe(CN)6and K4Fe(CN)6) was used above a raw electrode without aptamer. After our experiments the optimal concentration was found to be 5mM of each chemical in a 0.1 KCl solution.
Cover the electrode with a ferricyanide droplet and connect it to the potentiostat.
Run a preliminar cyclic voltammetry test with a stardart parameters. The ones that have fits better with our hardware (Rodeostat) was one cycle from -0.3V to 0.3V, with a current limit of 1000 uA, a sample rate of 100 Hz and a scan rate of 0.05 mV/s.
After the test have finished, adjust the parameters (voltage range and current limit) to fit the complete curve in your range. A typical Cyclic Voltammetry curve may have a shape similar to a duck.
Wash the electrodes three times with deionized water and let them dry under an extraction hood air flow.
To remove the excess of DNA, treat the electrodes with 10 uL of β-Mercaptoethanol for 50 minutes in a humidity chamber.
Wash the electrodes three times with deionized water and let them dry under an extraction hood air flow.
Selecting the electrode
There are so many scaffolds to join the Aptamers (or the DNA). Our choice was based on the kind of measuring hardware that we have used, a potentiostat. For this variety of measuring system you need a 3-electrodes system (working, reference and counter electrodes). The other parameters of the electrode was choose as follows:
Ordering the DNA
To run the first Proof of Concept we ordered a commercial Thrombin aptamer. Some tips have been took into account for the aptamer adaptation to electrode binding: