Agencourt ampure xp kit for dna purification

Libraries were amplified on an Applied Biosystems Veriti^™ 96 well Thermal Cycler (Thermo Fisher Scientific) in a total reaction volume of 130 μl, containing 100 μl of Platinum^®PCR SuperMix High Fidelity, 5 μl of Library Amplification Primer Mix (both provided by the IPFL kit) and 25 μl of unamplified library. The cycling program suggested on the IPFL kit protocol was applied. Amplified libraries were purified with Agencourt^® AMPure^® XP Kit for DNA purification (Beckman Coulter, Beverly, Massachusetts, USA) on a DynaMag^™-2 magnet magnetic rack (Thermo Fisher Scientific) following the procedure proposed by the manufacturer. Agilent 2100 Bioanalyzer (Agilent Genomics) was used to determine the molar concentration of each barcoded library. Three equimolar pools of barcoded libraries were prepared: barcoded libraries from SUR-1 to SUR-6 (Pool 1), from SUR-7 to SUR-12 (Pool 2) and from SUR-13 to SUR-16 (Pool 3) were pooled together. The three pools were quantified on Agilent 2100 Bioanalyzer (Agilent Genomics) or Library TaqManTM Quantitation Kit (Thermo Fisher Scientific) following the procedure proposed by the manufacturer, and then diluted as proposed by the Ion PGM^™ Hi-Q^™ Chef Kit (Thermo Fisher Scientific).

20 ng of each amplified sample were diluted in a total volume of 79 μl of nuclease-free water (Life Technologies). Amplicons’ end-repair was done by adding 20 μl of 5X End Repair Buffer and 1 μl of End Repair Enzyme (both provided by the IPFL kit) and incubating the reaction at room temperature for 20 minutes. The samples were then purified with Agencourt^® AMPure^® XP Kit for DNA purification (Beckman Coulter, Beverly, Massachusetts, USA) on a DynaMag^™-2 magnet magnetic rack (Thermo Fisher Scientific) following the procedure proposed by the manufacturer.

The DNA samples were amplified with the primer pairs 16sf-var 5´-CAAATTACGCTGTTATCCCTATGG-3´ and 16sr-var 5´-GACGAGAAGACCCTAATGAGCTTT-3´ designed by Chapela et al. [20 ] using illustra^™ puReTaq Ready-To-Go^™ PCR Beads (GE Healthcare). For each tube containing the bead, 2 μl of 200 nM of each primer, 1 μl of 50 ng of template DNA and nuclease-free water (Life Technologies) were added, for a final reaction volume of 25 μl. DNA was amplified on an Applied Biosystems Veriti^™ 96 well Thermal Cycler (Thermo Fisher Scientific) with the following cycling program: denaturation at 94°C for 3 min; 35 cycles at 94°C for 40 s, 60°C for 40 s, and 72°C for 40 s; final extension at 72°C for 7 min. 5 μL of each PCR product was checked by electrophoresis on a 2% agarose gel and the presence of fragments of the expected length was assessed by a comparison with the standard marker O'GeneRuler DNA Ladder (Thermo Fisher Scientific). Double-stranded PCR products were purified with Agencourt^® AMPure^® XP Kit for DNA purification (Beckman Coulter, Beverly, Massachusetts, USA) on a DynaMag^™-2 magnet magnetic rack (Thermo Fisher Scientific) following the procedure proposed by the manufacturer. Purified PCR products were quantified using a Qubit^™ 3.0 Fluorometer (Thermo Fisher Scientific).