PacBio Onso - Onso Clustering Reagent Plate Preparation

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PN 103-140-400 REV05 JUN2024
3b.5
Calculate the sample library volume required per lane as follows:
Calculating sample library volume (per lane)
i = (g * e)/h
User input
g
Final sample library concentration (pM)
h
Starting sample library concentration (pM)*
500
e
Final library volume (µL)
380
i
Volume of starting sample library (µL)
*Note: It is recommended to have sample library input concentration (h) of at least 500 pM, but this value
can be increased if the volume of LQC + DNA library > 16 µL.
3b.6
Dilute the sample library to a starting concentration of 500 pM using Low TE buffer for the required volume
(calculated in Step 3b.5) per lane. Vortex to mix and spin down.
If volume of starting sample library (from Step 3b.5) + volume of starting LQC library (from
Step 3b.3) exceeds 16 µL, it is recommended to adjust starting sample and/or LQC
concentrations to be > 500 pM.
3b.7
Prepare 100 µL of fresh 0.1N sodium hydroxide (NaOH).
3b.8
In a DNA low-bind tube, add the volume of starting sample library (i) and volume of starting LQC library (f),
per lane.
3b.9
Add an equivalent volume of fresh 0.1N NaOH as calculated in Step 3b.8 to the same tube. Gently pipette mix
up and down at least 10 times and spin down. Do not vortex. The volume of starting sample library (i) +
volume of starting LQC library (f) + 0.1N NaOH should not exceed 32 µL per lane (e.g., Add 10 µL of fresh
0.1N NaOH to 10 µL of combined (sample + LQC) libraries.)
3b.10
Incubate the library and NaOH mixture for 5 minutes at room temperature to allow for proper denaturation.
3b.11
Add DIL buffer to the library, LQC, and NaOH mixture to reach a final volume of 380 µL. Pipette mix and spin
down. Immediately proceed to section 4 titled “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 “Clustering reagent plate preparation”
4. Onso clustering reagent plate preparation

Step
Instructions
4.1
Puncture the foil seal in positions A01 and B01 with a sterile pipette tip and discard the used tip.
4.2
Puncture the oval foil seal in column 2 with a sterile pipette tip and discard the used tip.
4.3
Add 375 µL of diluted library to position A01 or B01 (A01=Lane 1, B01=Lane 2).
Leave the oval trough in column 2 empty until after the clustering run has completed (Refer to
section 5 “Post-run wash”).