Rotator with variable speed (15 rpm) for 1 ml tubes
Freezers (-20 and -80oC)
Centrifuge – 13000rpm
Buffers for SELEX
Binding buffer/Wash buffer:
DTT- Dithiothreitol, Tris-HCl pH 7.5
Tris-HCl (50mM) – 7.88g for 1L
NaCl (25mM) – 1.461g for 1L
MgCl (5mM) – 0.476g for 1L
DTT (10mM) – 1.5425g for 1L
Elution buffer:
Sodium acetate (0.4M) – 32.812g for 1L
EDTA (5mM) – 1.86g for 1L
Urea (7M) pH 5.5 – 420.42g for 1L
Ammonium acetate (7.5M) – 578.7g for 1L/ 28.9g for 50ml
Procedure
Pre-wetted
Soak membrane in sterile water for 1min and put membrane in the filter holder;
To eliminate DNAs that bound to the membrane, DNAs (1 μg/μl) were passed three times prior to the selection cycle through pre-wetted nitrocellulose acetate membranes in “Pop-top” filter holders;
DNA denature
For the first cycle of selection, 35.5 μl (1μg/ μl) DNAs were added in 114.5 μl binding buffer solution (volume 150 μl);
And then were denatured at 95oC for 10 min;
Then were allowed to cool in thermocycler to approximately 300C (about 1.5 hours);
Binding
Denatured DNAs (35.5 μl, 1μg/ μl DNAs + 114.5 μl binding buffer) + 50 μl target protein (87 μg/ml) were incubated at 15 rpm on a variable speed rotor for 1 hour and 45 min at room temperature.
Washing
Soak membrane in wash buffer (the same as binding buffer) for appr. 1 min and then put the membrane in the filter holder;
Aspirate the binding reaction mixture (DNAs+target) with a syringe with a needle
Remove the needle and inject the binding reaction mixture into the filter holder with a membrane and then inject 1 ml wash buffer solution (the same as binding buffer) and let wash buffer pass through the membrane.
Elution
Heat 200ul elution buffer(0.4 M sodium acetate, 5 mM EDTA, 7M urea, pH 5.5) to 70-800C
Inject 200 μl of elution buffer into the filter holder to elute DNA that was retained in the filter into a fresh tube. Make sure that all liquid is out.
Take filter and cut into 4 pieces and put into elution buffer with DNA and put the tube to water bath at 70-80oC over the course of 5 min.
The elute was transferred to a fresh tube (Elute 1) and filter was eluted again as above (Elute 2)
Precipitation
The elutes (1 and 2) were diluted with 200 μl H2O (each) before ethanol precipitation;
For precipitation: 2x6 μl glycogen (20 mg/ml) + 2x200 μl ammonium acetate (7.5 M) + 2x1 ml ethanol (100%) were incubated for 1 hour (or can leave overnight if no pellet is observed after centrifugation) at -80oC;
Centrifugation under 13,000 rpm at 4oC for 1 hour;
Carefully remove the supernatant without disturbing the pellet (S1 and S2 – keep supernatant just in case)
Washing the pellet with 75% ethanol solution (-20oC) (1000 μl) (add 1000 ul of ethanol and make sure the pellet came out; centrifuge for 5 min to let the pellet settle again; remove ethanol without disturbing the pellet leaving some liquid in the tube)
Repeat previous step one more time (2 washes of each pellet; WP1-1, WP1-2 for pellet from E1; WP2-1, WP2-2 for pellet from E2)
After the second wash carefully remove ethanol without disturbing the pellet with a pipette
Dry under the current of air in horizontal form for 2-3 minutes until it becomes transparent (maximum 5 minutes)
Resolve the pellet of Elute 1 in 50 μl pure water
After washing and drying the pellet from Elute 2, add resolved pellet from Elute 1 (50ul) into it and it is ready to do PCR.
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