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.

  

  3. Separate the 3D printed structures from the printer base. Remove the excess of printed material.

  4. Treat the columns with dichloromethane until the surface gets smooth.

  

  ADVICE: For us it have worked putting the columns in glass jar, above a cardboard pedestal. Then cover the base of the jar with dicloromethane without touching the 3D printed files. Put the jard on the 3D printed hotbed at 80ºC for 20 minutes.

  5. Wash the columns three times in deionized water to clean them from dicloromethane.

  6. 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.

  7. Keep in milliQ water until its use.

  Designing and ordering the initial library

  Amount of time: 20 mins

  Total costs: 100 € (Without been sponsored)

  8. 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.

  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

  9. 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).

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

  11. 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.

  CAUTION: The colums break easily, so do not aplyy too much force on them.

  12. 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

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

  14. Apply the DNA to a new nitrocellulose membrane as in step 11.

  15. Wash the membrane four times with 300 µl of BB, like on step 11.

  16. Recover the membrane and transfer it to a new Eppendorf tube.

  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

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

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

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

  20. 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.

  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

  21. 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.

  22. 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:

  

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

  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.

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

  25. 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.

  PAUSE POINT:The library can be stored at -20ºC

  CAUTION: 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:

  26. 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.

  27. Perform the PCR amplification with the same condition as step 22 and take the samples at the specified cycles

  28. Prepared an agarose gel at 3%.

  29. Perform the electrophoresis gel at 90V for 50 min.

  30. Select the maximum number of cycles where you can a see a clear band without unspecific products.

  PAUSE POINT: You can store the DNA at -20ºC

  ADVICE: 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:

  31. Prepare a 200 µL PCR. Use as template 2 µL of the library amplified before and a final primer concentration of 0.8 µM..

  32. Use the same programme but with the cycles chosen before

  33. Perform a new electrophoresis gel to ensure that the amplification was successful. Purified the DNA and stored it at -20ºC.

• PCI

  PCI

• Purification

  Purification

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Aptamer Characterization

Elona