Luminex 100 IS Version 2.1 - Manual (Page 78)

Luminex 100 IS Developer Guide to xMAP Technology Version 2.1 x MAP Technology
72 PN 89-00002-00-062 Rev. A
4. Remove the supernatant, being careful not to disturb the pellet.
5. Add 50 µL of 0.1 M MES (pH 4.5). Vortex and sonicate.
6. Add 0.2 nmole of amino-substituted oligonucleotide (that is, 2
µL of a 1:10 dilution of the 1 mM solution). Vortex briefly (See
Technical note 3).
7. Immediately before use, prepare a 10 mg/mL solution of EDC
powder (See Technical note 4). Vortex until dissolved.
8. Add 2.5 µL of the fresh EDC solution to the microspheres.
Vortex immediately.
9. Incubate for 30 minutes at room temperature in the dark.
10. Repeat steps 7 - 9 with fresh EDC (for a total of two EDC
additions).
11. Add 1.0 mL of Tween 20 (0.02% v/v). Vortex.
12. Microcentrifuge microspheres at ≥ 8,000 × g for 1 minute.
13. Remove the supernatant, being careful not to disturb the pellet.
14. Add 1.0 mL of SDS (0.1% w/v). Vortex.
15. Microcentrifuge the microspheres at ≥ 8,000 × g for 1 minute.
16. Remove the supernatant, being careful not to disturb the pellet.
17. Resuspend the microspheres in 100 µL of TE (10mM Tris, 1mM
EDTA, pH 8.0).
18. Enumerate the microsphere preparation.
19. Store the preparation at 2
o
C - 8°C. Protect it from light.
Technical Notes
1. Oligonucleotides must be synthesized with a 5’ or 3’ amino
group. No Tris or Azide or other amine-containing buffers
should be present during the coupling procedure. Preferably, the
oligonucleotide should be resuspended in water following
synthesis. If oligonucleotides were previously solubilized in an
amine-containing buffer, then precipitation and resuspension into
water is required.
2. This procedure can be scaled up or down.