Bio-Rad Nonmagnetic Beads and Related Reagents - Manual (Page 26)

9.2 Sample Protocol Using Bio-Plex COOH Beads
Notes:
a. Beads must be completely protected from light throughout this
procedure.
b. The stock bead concentration is 1.25 x 10 7 beads /ml.
c. 1x scale = 1.25 x 10 6 beads.
d. Volume stock beads required (ml) = [(1.25 Ч 10 6 ) Ч (scale)] ч [1.25 Ч
10 7 bead/ml]
Procedure:
1. Bring a fresh aliquot of –20°C desiccated EDAC powder to room
temperature.
2. Resuspend the amine-substituted oligonucleotide (probe or capture
oligo) to 1 mM (1 nmole/µl) in dH
2
O.
3. Resuspend the stock vial of beads by vortexing at speed 7, followed
by sonication for 15 sec.
4. Transfer desired volume of the stock beads (based on the scale) to an
appropriately sized tube. For 1–25× scale, use a 1.5 ml siliconized
clear microcentrifuge tube.
5. Pellet the beads by microcentrifugation at ≥ 8000 x g for 1–2 min.
6. Carefully remove the supernatant without disturbing the beads.
7. 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.
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