Phix174 rf 2 dna

The unique index sequence of the ETS amplicons was generated as described previously (Quail et al., 2014 (link)). ETS amplicons were prepared by PCR using PhiX174 RF II DNA (New England Biolabs) as template DNA. In brief, each amplification reaction consisted of 200 ng of PhiX174 DNA (New England Biolabs), 0.5 µM of ETS-indexes forward primer, 0.5 µM ETS universal reverse primer and Q5 hot start high-fidelity 2× master mix (New England Biolabs). PCRs were performed on a Labcycler thermal cycler (Sensoquest GmbH) using the following conditions: 98°C for 30 s, 35 cycles of 98°C for 10 s, 60°C for 20 s, 72°C for 30 s and a final elongation step at 72°C for 30 s. A total of three PCR reactions were performed for each ETS amplicon. PCR products of each ETS amplicon were pooled and purified using the QIAquick PCR Purification Kit (Qiagen) and eluted in 30 µl of Qiagen elution buffer. Fragment concentration was measured using the Qubit™ dsDNA HS Assay Kit (Invitrogen). Each ETS amplicon was adjusted to a final concentration of 3 ng/µl, aliquoted and stored at −20°C as stock ETS plates. ETS amplicons were diluted further to 0.03 ng/µl. ETS fragments (Fig. 1a) and the entire ETS design (Fig. 1, Supplementary Table SI) were optimized and validated for sequencing-based PGT.