Bio-Rad Nonmagnetic Beads and Related Reagents - Manual (Page 23)

Section 9
Coupling of Amine-Modified
Oligonucleotides
Bring all the buffers to room temperature prior to use. Protect the beads
from light as much as possible by covering the tubes with aluminum foil
during the procedure. Remove EDAC from the –20ºC freezer and store them
in a desiccator at room temperature for approximately 1 hr prior to their use.
The bottle of EDAC and should be discarded after five uses.
9.1 Sample Protocol Using Bio-Plex Pro Magnetic 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]
e. Capture oligo concentration should be optimized for the specific
reagents in use. Typically, optimal coupling is achieved using 0.2 to 1
nmole per scale.
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.
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