Bio-Rad Nonmagnetic Beads and Related Reagents - Manual (Page 24)

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5. Place the tube into a magnetic separator and allow separation to occur
for 30–60 sec.
6. With the tube still positioned in the magnetic separator, carefully remove
the supernatant without disturbing the beads.
7. Remove the tube from the magnetic separator and resuspend the
beads in 50 µl of 0.1 M MES, pH 4.5, by vortex and sonication for
approximately 20 sec.
8. Prepare a 1:10 dilution of the 1 mM capture oligo in dH
2
O (0.1
nmole/µl).
9. Add 2 µl (0.2 nmole) of the 1:10 diluted capture oligo to the
resuspended beads and mix by vortexing.
10. Prepare a fresh solution of 10 mg/ml EDAC in dH
2
O.
11. Add 2.5 µl of fresh 10 mg/ml EDAC to the beads (25 µg or ≅ [0.5
µ
g/µl]final) and mix by vortexing.
12. Incubate for 30 min at room temperature in the dark.
13. Use a fresh aliquot of EDAC powder to prepare a second fresh solution
of 10 mg/ml EDAC in dH
2
O.
14. Add 2.5 µl of fresh 10 mg/ml EDAC to the beads and mix by vortexing.
15. Incubate for 30 min at room temperature in the dark.
16. Add 1.0 ml of 0.02% Tween-20 to the coupled beads (use the same
volume for 1–25x scale).
17. Place the tube into a magnetic separator and allow separation to occur
for 30–60 sec.
18. With the tube still positioned in the magnetic separator, carefully remove
the supernatant without disturbing the beads.
19. Remove the tube from the magnetic separator and resuspend the
coupled beads in 1.0 ml of 0.1% SDS by vortex (use the same
volume for 1–25x scale).