Access Array System for the Ion Torrent PGM Sequencing System: User Guide
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Chapter 6: Post-PCR Amplicon
Purification and Quantitation
This chapter describes a standard Access Array™ procedure for the analysis of PCR products
harvested from an LP 48.48 IFC.
The quality of the PCR products prepared on an LP 48.48 IFC (or a Access Array 48.48 IFC) is
critical for successful amplicon sequencing. Any contamination of primers or primer dimers in
the PCR products will be directly reflected in the quality of sequencing reads. Therefore, the
PCR products generated on an IFC should to be qualified and purified before sequencing.
The PCR products generated on the IFC are first analyzed using an Agilent® 2100
Bioanalyzer to check for quality. Next, the PCR products are pooled together in equal volume
to create one PCR product library. The PCR product library is then purified using AMPure XP
beads, and quantified before proceeding to sequencing. It is recommended to use the
Quant-iT PicoGreen dsDNA assay kit for quantification of the final (cleaned-up) library prior to
sequencing.
Reference Documents
•
Agilent
® DNA 1000 Kit Guide
•
Quant-iT™ PicoGreen® User Guide
Materials
Required Equipment
•
SPRIPlate
®
96R Magnet Plate (Agencourt, PN 000219)
or DynaMag™-2 magnet (Invitrogen, PN 123-21D)
•
Agilent 2100 Bioanalyzer and DNA 1000 Kit (Agilent, PN 5067-1504)
•
Fluorimeter-compatible 96- or 384-well microtiter plates
Required Reagents
Stored at 4 °C
•
Agencourt AMPure XP Reagent beads (Beckman Coulter Genomics, PN A63880)
•
Quant-iT
PicoGreen
dsDNA Assay Kit (Invitrogen, P11496)
