Bio-Rad Nonmagnetic Beads and Related Reagents - Manual (Page 27)

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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. Pellet the beads by microcentrifugation at ≥ 8000 x g for 1–2 min.
18. Carefully remove the supernatant without disturbing the beads.
19. Resuspend the coupled beads in 1.0 ml of 0.1% SDS by vortex (use
the same volume for 1–25x scale).
20. Pellet the beads by microcentrifugation at ≥ 8000 x g for 1–2 min.
21. Carefully remove the supernatant without disturbing the bead pellet.
22. Resuspend the coupled beads in 100 µl of TE, pH 8.0, by vortex and
sonication for approximately 20 sec.
23. Enumerate the coupled beads by hemacytometer:
a. Dilute the resuspended coupled beads 1:100 in dH
2
O.
b. Mix thoroughly by vortex.
c. Transfer 10 µl to the hemacytometer.
d. Count the beads within the 4 large corners of the
hemacytometer grid.
e. Beads/µl = (sum of beads in 4 large corners) x 2.5 x 100
(dilution factor).
f. Note: maximum is 50,000 beads/µl.
24. Store coupled beads refrigerated at 2–8°C in the dark.