Team:Madrid-OLM/AptamerProtocols
Aptamer's Protocols
Texto de explicacion/ resumen de la pagina.
Aptamer Discovery
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SELEX
SELEX
Bill Of Materials: You could see a complete BoM here (upload the bill).
Amount of time: 1 day
Total costs: 100 €.
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Download the columns of the stl files from our github repository.
3D print the stl models in PETG. For more information about the reasons why we choose this material see the results page.
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Separate the 3D printed structures from the printer base. Remove the excess of printed material.
Treat the columns with dichloromethane until the surface gets smooth.
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Wash the columns three times in deionized water to clean them from dicloromethane.
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Put the columns in sterilizing solution (0,1N NaOH, 1% (m/v) EDTA) to inactivate DNAses and remove other pollutants. Keep overnight at room temperature.
Keep in milliQ water until its use.
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Design your library as a DNA of 30-40 random nucleotides flanked by constant extremes of 12-18 nucleotides. Use HPLC purification. Also order the primers for this constant edges.
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Resuspend 2 nmol de la library pool on 200 µl of Binding Buffer (Tris-HCl PH= 7,4 20 mM; MgCl21mM; NaCl 150mM; KCl 5 mM).
Denatured the library by heating it at 90ºC for 10 min and immediately cold it on ice for another 10 min.
Wash in distilled water and mount the nitrocellulose column by cutting a small square of the membrane and then pre-wet it with the BB.
To get rid of the DNA that unespecifically binds to the system, apply the library through a nitrocellulose membrane and centrifuge 1 min at 8000 rpm. Quantify the DNA that does not bind unspeficically and note it as the initial DNA.
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Incubate the flowthrough with the protein of interest during 1 hour.
Apply the DNA to a new nitrocellulose membrane as in step 11.
Wash the membrane four times with 300 µl of BB, like on step 11.
Recover the membrane and transfer it to a new Eppendorf tube.
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Add 400µL of FES and 500 µL of phenol and mix in a thermomixer/ vortex at 1.400 rpm for 10 min.
Transfer the liquid to a new tube and repeat step 8 but this time with 200 µl of each regeant.
Mix the two samples and add 200 µl of Milli-Q wáter to allow the phase separation and centrifuge 10 min at 16100 g.
Transfer the aqueous phase (upper) to a new 2 ml tube and made a PCI or Qiagen (link) columns to extract the DNA. Resuspend the purified DNA in 30 ul of Milli-Q water.
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Prepare the PCR mixture for a final volume of 50 µl per reaction and a final primer concentration of 0,8 µM. For the first round use all the template recover after the incubation. For the next rounds use 20 ul of template and adjust the rest according to the reagent you use.
Perform the amplification with the following amplification conditions. Adjust the annealing temperature according to the primers used, and the hotstart to the specifications of your polymerase:
Prepare an agarose gel at 3%. Load the samples and perform the electrophoresis at 90V for 50 min.
Remove the gel and observe the bands under UV light.
It is needed at least 1 ug to continue with the next round. If it not accomplish, a further amplification is needed (continue reading). If you succeed amplifying with 10 cyclis this amount of DNA, skip the next steps and continue repeating this steps to do the next SELEX round.
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The total PCR reaction mixture volume for each tube is 50 µl using as template 0,5 µl of the library amplified before, for each tube, and a final primers concentration of 0.8µM. Choose PCR samples at the following cycles:5, 10, 15, 20, 25. Also a negative control tube at the twentieth cycle.
Perform the PCR amplification with the same condition as step 22 and take the samples at the specified cycles
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Prepared an agarose gel at 3%.
Perform the electrophoresis gel at 90V for 50 min.
Select the maximum number of cycles where you can a see a clear band without unspecific products.
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Prepare a 200 µL PCR. Use as template 2 µL of the library amplified before and a final primer concentration of 0.8 µM..
Use the same programme but with the cycles chosen before
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Perform a new electrophoresis gel to ensure that the amplification was successful. Purified the DNA and stored it at -20ºC.
DIY nitrocellulose column manufacture
ADVICE : We have found the following parameters as the optimal ones printing with a Prusa i3 machine:-Filaments diameter of 1.75mm
-Nozzle at 230ºC. Base 80ºC with Nelly hairspray. (CAUTION: The brand of the headspray must be Nelly.
ADVICE : For us it have worked putting the columns in glass jar, above a cardboard pedestal. Then cover the base of the jar with dichloromethane without touching the 3D printed files. Put the jard on the 3D printed hotbed at 80ºC for 20 minutes.Designing and ordering the initial library
ADVICE : For us, IDT have worked well as a DNA provider. They are also iGEM sponsor at our year, so this libraries could be free for igem teams.ADVICE : The following sequence have fit well to us:
Prepare the library pool
CAUTION : The colums break easily, so do not aplyy too much force on them.Protein-Aptamer incubation
CAUTION : Do not let the membrane dry, as it becomes fragile and the mollecules inside it could be damage.Denatured the protein and elute the selected DNAs
ADVICE : Qiagen colums recover more DNA and also reduced the time of the purification, but are more expensive.PAUSE POINT :You can leave the PCI precipitation overnight (see PCI protocol), or the Qiagen Purified DNA in the fridge at 4ºC.Library amplification
ADVICE : We strongly recommend to quantify the DNA by gel molecular mass marker instead other methods like nanodrop. Add in this step to the first line of your gel if you decide to use this method.ADVICE : For revealing the gel bands, GelRed have fits correctly to our purpose. We have put the GelRed before the gel polymerization step inside the mixture, following the product specifications.PAUSE POINT :The library can be stored at -20ºCCAUTION : We strongly recommend you to keep a little portion of each round of selection as a backup plan in case that you lost your DNA in further rounds. Keep in mind this when you amplify your DNA, because you will need more that the 1ug of DNA used in the next SELEX round.Determination the optimal number of amplification cycles:
PAUSE POINT : You can store the DNA at -20ºCADVICE : If you always have secondary bands, it means that concatemers are forming in your PCR reactions. Consider reducing the template and/or the cycles you are performing.ADVICE : Select the rounds that have the maximum amount of DNA that fits to your needs without secondary bands. Its more important to have the correct purity if you already are going to have the necessary amount. If secondary structures are always forming in your PCR, consider purifying the correct bands from your gel with a kit..Preparative PCR:
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Manual PCI Purification
PCI Extraction and ethanol precipitation
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Add an equal volume of PCI (phenol: chloroform: isoamyl alcohol 25:24:1) to the digested DNA solution to be purified in a 1.5-ml microcentrifuge tube.
Mix gently for 5 min (rocking platform or vortex) and microcentrifuge 10 min at 10,000 rpm at room temperature.
Remove the top (aqueous) phase containing the DNA and transfer to a new tube. Repeat steps 1-3.
Add an equal volume of CI ( chloroform: isoamyl alcohol 24:1). Mix gently for 2 min and centrifuge for 1 min at 10,000
Remove the top (aqueous) phase containing the DNA and transfer to a new tube.
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Add 3 volumes of ice-cold 100 ethanol and 1/10 volumes of 3M Sodium acetate. Invert the tube and place in -20 ºC overnight or in -70ºC for 1 hour.
Spin 30 min in a fixed-angle microcentrifuge at 16 100g and 4ºC. Remove the supernatant.
Add 1 ml of room-temperature 70% ethanol ( if the DNA molecules are very small, less than 200 pb, use 95% ethanol) and only wash the pellet. microcentrifuge as in step 2.
Spin 10 min at 16 100g and remove the supernatant
Let the pellet air dry for 20 min.
PCI Extraction:
CAUTION : work with all the reagents in an extration hood.Ethanol Precipitation:
CAUTION : Wash the pellet carefully. Invert the tube gently. -
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Quiagen Purification
Quiagen Purification
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Add 5 volumes of Buffer PB to 1 volume of the PCR sample, and then mix. It is not necessary to remove mineral oil or kerosene. For example, add 500 μl of Buffer PB to 100 μl PCR sample (not including oil).
If pH indicator I has been added to Buffer PB, check that the mixture’s color is yellow. If the color of the mixture is orange or violet, add 10 μl of 3 M sodium acetate, pH 5.0, and mix. The color of the mixture will turn yellow.
Place a QIAquick spin column in a provided 2 ml collection tube.
To bind DNA, apply the sample to the QIAquick column and centrifuge for 30–60 s.
Discard flow-through. Place the QIAquick column back into the same tube. Collection tubes are reused to reduce plastic waste.
To wash, add 0.75 ml Buffer PE to the QIAquick column and centrifuge for 30–60 s.
Discard flow-through and place the QIAquick column back into the same tube. Centrifuge the column for an additional 1 min
Place QIAquick column in a clean 1.5 ml microcentrifuge tube.
To elute DNA, add 50 μl Buffer EB (10 mM Tris•Cl, pH 8.5) or water (pH 7.0–8.5) to the center of the QIAquick membrane and centrifuge the column for 1 min. Alternatively, for increased DNA concentration, add 30 μl elution buffer to the center of the QIAquick membrane, let the column stand for 4 min, and then centrifuge.
CAUTION : Residual ethanol from Buffer PE will not be completely removed unless the flow-through is discarded before this a Ensure that the elution buffer is dispensed directly onto the QIAquick membrane for complete elution of bound DNA. dditional centrifugationCAUTION : Residual ethanol from Buffer PE will not be completely removed unless the flow-through is discarded before this a Ensure that the elution buffer is dispensed directly onto the QIAquick membrane for complete elution of bound DNA. dditional centrifugation -
Back to Dicovery Protocol Index
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
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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).
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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.
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[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.
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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:
Aptamer Bounding
Electrodes testing with cyclic voltammetry
