Team:Madrid-OLM/AptamerProtocols
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 €.
-
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.
-
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.
-
Wash the columns three times in deionized water to clean them from dicloromethane.
-
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.
-
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.
-
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.
-
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.
-
Add 400µL of FES and 500 µL of phenol and mix in a thermomixer at 1.400 rpm for 10 min.
Transfer the liquid to a new tube and repeat the 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 PCI extract the DNA.
-
Prepare the PCR mixture for a final volume of 50 µl per reaction and a final primer concentration of 0,8 µM.
Perform the amplification with the following amplification conditions (adjust according to the primers used):
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.
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 dicloromethane 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
Amount of time: 20 mins
Total costs: 100 € (Without been sponsored)
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
PAUSE POINT : You can leave the PCI precipitation overnight.Library amplification
-
-
PCI
PCI
-
Purification
Purification
Back to Dicovery Protocol Index