illumina cBot, System Manual

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cBot 2 System Guide
21
}
Laboratory-grade water (900 µl)
} Stock 1 N NaOH (100 µl)
2 Invert to mix.
Denature Libraries and Add Optional PhiX Control
The library loading concentration depends on the libraries to be sequenced. The following
instructions apply to either TruSeq Nano DNA (350 bp) or TruSeq DNA PCR-Free (350 bp)
libraries. Dilute to a concentration appropriate for the library type.
}
A DNA loading concentration that is too high causes reduced %PF.
}
A DNA loading concentration that is too low causes reduced %PF and a high
percentage of duplicates that negatively affect depth of coverage.
Repeat the following instructions for each flow cell to be sequenced.
1 Dilute the library or pooled libraries to the appropriate concentration:
}
TruSeq Nano DNA libraries —Dilute to 2–3 nM in RSB.
} TruSeq DNA PCR-Free libraries —Dilute to 1–2 nM in RSB.
2 [Optional] Spike in 1% nondenatured Illumina PhiX control to nondenatured libraries:
}
TruSeq Nano DNA libraries —Add 0.5 µl 2–3 nM PhiX to 50 µl 2–3 nM library.
}
TruSeq DNA PCR-Free libraries —Add 0.5 µl 1–2 nM PhiX to 50 µl 1–2 nM library.
3 Number the tubes of an 8-tube strip:
}
For clustering with sample tracking—From the key end, label the tubes #8 through
#1.
}
For clustering without sample tracking—Label the tubes #1 through #8. When
preparing 4 flow cells, consider adding another designator on the 8-tube strip for
proper tracking.
4 Denature the library in the 8-tube strip as follows:
a Add 5 µl nondenatured library to the bottom of each well.
b Add 5 µl freshly diluted 0.1 N NaOH. Slowly pipette to mix.
c Incubate at room temperature for 8 minutes.
d Add 5 µl 200 mM Tris-HCl pH 8.0. Slowly pipette to mix.
5 Set aside on ice until you are ready to add the ExAmp master mix.
CAUTION
Prepare and add the ExAmp master mix within 30 minutes .
Prepare the cBot Reagent Plate
1 Invert to mix.
2 Vortex to dislodge trapped air bubbles.
3 Tap on a hard surface to collect reagent droplets. Alternatively, pulse centrifuge.
4 Set aside on ice.