Guidelines For Library Dilution; Dilute The Sample Library; Dilute The Control Library; Prepare The Consumables - Thermo Scientific Ion 520 User Manual

Guidelines for library dilution

• The sample library dilution recommendations below represent optimal input concentrations for
control libraries. Users who have already optimized a library concentration for other Ion Chef
reagent kits or the manual Ion PGM
concentration for the Ion 520
• Recommendations are based on qPCR quantification. If libraries are quantified with an Agilent
2100 Bioanalyzer
equivalent input.
• Use polyclonality and low-quality filter results from a sequencing run performed with ISPs
templated at the starting concentration, then titrate up or down to achieve optimal concentrations,
if needed.
• Prepare a fresh dilution of each library before use with the Ion Chef
dilutions within 48 hours.
• Barcoded libraries are typically combined (or pooled) before templating to allow multiple libraries
to be sequenced on a single chip. We recommend that you combine equal volumes of individual
barcoded libraries at the same concentration (for example, 100 pM), then dilute the combined
library to a concentration recommended in the following table.

Dilute the sample library

IMPORTANT! Before proceeding, dilute the library or combined library to the optimal input
concentration. The quality of your sequencing data relies on achieving the correct concentration of
starting library.
Dilute the individual or combined library according to the following table.
Library read length
400−600 bp

Dilute the control library

If you are running a control library for troubleshooting purposes, dilute the appropriate library obtained
from one of the controls kits, according to the following table:
Control library
E. coli DH10B Control 400 Library
E. coli DH10B Control 600 Library

Prepare the consumables

1. Unbox the Ion S5
room temperature.
IMPORTANT! The Reagents cartridge must sit at room temperature for 45 minutes before use.
Ion 520 ™ & Ion 530 ™ ExT Kit − Chef User Guide
™
Template IA 500 Kit may need to re-optimize library
™ ™
& Ion 530
™
instrument, a higher calculated concentration may need to be used for
Recommended concentration
Volume of library
™
ExT Chef Reagents cartridge 45 minutes before use, then allow it to warm to
Chapter 4 Start the Ion Chef
ExT Kit – Chef.
40–100 pM
2 µL
2 µL
™ run

Prepare the consumables

™
System, and use the library
Molecules per input volume
9
1.8−3.0 × 10
Volume of Nuclease-free Water
48 µL
48 µL
4
™
™
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