Team:Madrid-OLM/AptamerProtocols

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Aptamer's Protocols

Texto de explicacion/ resumen de la pagina.

Aptamer Discovery

• SELEX

  SELEX

  Bill Of Materials: You could see a complete BoM here (upload the bill).

  Amount of time: 1 day

  Total costs: 100 €.

  DIY nitrocellulose column manufacture

  1. Download the columns of the stl files from our github repository.

  2. 3D print the stl models in PETG. For more information about the reasons why we choose this material see the results page.

  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.

  

  Prepare the library pool

  3. Resuspend 2 nmol de la library pool on 200 µl of Binding Buffer (BB).

  4. Denatured the library by heating it at 90ºC for 10 min and immediately cold it on ice for another 10 min.

  5. 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.

  Protein-Aptamer incubation

  6. Incubate the flowthrough with the protein of interest during 1 hour.

  7. Apply the DNA to a new nitrocellulose membrane as in step 3.

  8. Wash the membrane four times with 300 µl of BB, like on step 3.

  9. Recover the membrane and transfer it to a new Eppendorf tube without letting it dry.

  Denatured the protein and elute the selected DNAs

  10. Add 400µL of FES and 500 µL of phenol and mix in a thermomixer at 1.400 rpm for 10 min.

  11. Transfer the liquid to a new tube and repeat the step 8 but this time with 200 µl of each regeant.

  12. Mix the two samples and add 200 µl of Milli-Q wáter to allow the phase separation and centrifuge 10 min at 16100 g.

  13. Transfer the aqueous phase (upper) to a new 2 ml tube and PCI extract the DNA.

  PAUSE POINT: You can leave the PCI precipitation overnight.

  Library amplification

  14. Prepare the PCR mixture for a final volume of 50 µl per reaction and a final primer concentration of 0,8 µM.

  15. Perform the amplification with the following amplification conditions (adjust according to the primers used):

  

  16. Prepare an agarose gel at 3%. Load the samples and perform the electrophoresis at 90V for 50 min.

  17. Remove the gel and observe the bands under UV light.

  18. It is needed at least 1 ug to continue with the next round. If it not accomplish , a further amplification is needed.

• PCI

  PCI

• Purification

  Purification

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Aptamer Characterization

Elona