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PN 103-140-400 REV05 JUN2024
Clustering procedure
1. Thawing clustering reagents
Each clustering reagent plate supports clustering of up to 2 lanes on a single flow cell.
Remove the Onso clustering reagent kit from -20°C storage, unbox and remove clustering reagent plate from
mylar bag. Discard mylar bag.
Thaw contents of the Onso clustering reagent kit:
• Place the DIL at room temperature to thaw. DIL can be stored at 4°C after thaw for future use.
• Allow clustering reagents plate to thaw for 1 hour in a room temperature (18-25°C) water bath. Water
should not pass more than halfway up the clustering plate (1.5 in.).
Thawing of DIL and clustering reagent plate can be conducted simultaneously.
After 1 hour thawing, remove the reagent plate from the water bath. Wipe the clustering reagent plate top and
underside dry.
Mix reagents in the clustering reagent plate by inverting the reagent pack slowly 10 times.
Gently tap the reagent plate on the benchtop 10 times to consolidate any reagent liquid to the bottom.
2. Preparing instrument wash reagent
The instrument wash reagent detailed here can be stored up to 2 weeks at 4°C. The preparation procedure detailed
below is for a final volume of 1 liter and makes use of a 10% (w/v) Tween-20 solution.
The final formulation of wash buffer contains 0.05% Tween-20, 10 mM Tris, 1 mM EDTA, and has a pH of 8.0.
The instrument wash reagent is required during the cluster generator pre-run checks, cluster generator post-run
wash, as well as when the cluster generator instrument requires a standby wash, or maintenance.
In a 1000 mL bottle, add 985 mL of molecular biology grade water.
Add 5 mL of a prepared 10% (w/v) Tween-20 solution to the bottle for a final concentration of 0.05% Tween-
20.
Add 10 mL of 100X TE buffer into the bottle.
Cap and invert the bottle at least 10 times to mix.
3. Denaturing and diluting the DNA library
Optimal loading concentration of your library for cluster generation will depend on your sample type and library
prep method. If not using a control library (LQC) spike-in, proceed with Step 3a.1. If using an LQC spike-in, proceed
with Step 3b.1. Refer to “Library loading concentration and control spike-in guidelines” under the section “General
best practices” for recommendations on loading concentrations and LQC spike-in percentages.
Combined volume of library and 0.1N NaOH should not exceed 32 µL to minimize the concentration of NaOH
carried over into the clustering reaction. If the desired loading concentration requires a library volume > 16 µL, it
is recommended to dilute your library to an input concentration > 500 pM.
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