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PN 103-140-400 REV05 JUN2024
All steps below should be duplicated to prepare both lanes of a single flow cell for clustering. Alternatively, if
clustering only one lane of the flow cell, perform instructions below for the 1 lane which will contain clustered
library, while skipping forward to step 3a.8 or 3b.11 guidance for the empty lane. Note that once a flow cell is
clustered once, it should not be re-clustered.
Users should perform theoretical calculations with the appropriate procedure in section 3 (either 3a or
3b) to first determine the library input volume required per lane BEFORE diluting library input
concentrations to 500 pM to ensure that minimum volume requirements are met.
3a. Denature and dilute DNA library WITHOUT LQC control library spike-in
Retrieve a 1 mL tube of DIL buffer from 4°C storage.
Calculate the library input volume required per lane as follows:
Calculating library input volume (per lane)
Desired total loading concentration (pM)
Starting library input concentration (pM)*
Final library volume (µL)
Volume of starting library (µL)
* Note: If the “Desired total loading concentration” (a) requires a “Volume of starting library” (d) > 16 µL, it is
recommended to change “Starting library input concentration (pM)” (b) to > 500pM.
Dilute the library to an input concentration of 500 pM using 10 mM Tris, 0.1 mM EDTA, pH8.0 for the required
volume (calculated in Step 3a.2-line d) per lane. Pipette mix and spin down.
If the “Desired total loading concentration” requires a library volume > 16 µL, it is recommended
to change “Starting library input concentration (pM)” (b) to > 500pM.
Prepare 100 µL of fresh 0.1N sodium hydroxide (NaOH).
For each lane to be clustered, obtain a new DNA low-bind tube, and add the required volume of starting library
(calculated in Step 3a.2 line d) for each respective lane. If using the same preparation in both lanes of the flow
cell, the reactions can be pooled into one tube.
Add an equivalent volume of fresh 0.1N NaOH as calculated in Step 3a.5 above to the respective tube
containing library. Pipette mix up and down at least 10 times and spin down. Do not vortex. The total volume
of library and 0.1N NaOH should not exceed 32 µL. (e.g. Add 10 µL of fresh 0.1N NaOH to 10 µL of library.)
Incubate the library and NaOH mixture for 5 minutes to allow for proper denaturation.
Add DIL buffer to the library and NaOH mixture to reach a final volume of 380 µL. Pipette mix and spin down.
Immediately proceed to section 4 titled “Onso clustering reagent plate preparation”.
If clustering a flow cell with an empty lane, simply add 380 µL of DIL buffer to a DNA low-bind tube designated
for the empty lane. Immediately proceed to section 4 titled “Onso Clustering reagent plate preparation”
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