Dna clean concentrate kit

Genomic DNA was extracted from the young leaf tissue using DNeasy plant Mini Kit (Qiagen) following the manufacturer's instruction. PCR amplification was performed using iProof High‐Fidelity DNA Polymerase (Bio‐Rad) with gene or allele‐specific oligonucleotide primers. The PCR conditions were as follows: initial denaturation at 98 °C for 5 min, 35 cycles of denaturation at 98 °C for 15 s, annealing and extension at ≥58 °C for 15 s, final extension at 72 °C for 15–30 s/kb. The annealing temperature for allele‐specific amplification was 63 °C (allele I) and 65 °C (allele II). Amplification products were analysed by agarose gel electrophoresis, purified using the DNA Clean & Concentrate Kit (Zymo Research) and sent for sequence analysis (Macrogen, www.macrogen.com). For subsequent verification, amplification product were cloned into pJET1.2/blunt cloning vector supplied in the CloneJET PCR cloning kit (Thermo Fisher Scientific) and at least four clones of each line were subjected to sequence analysis. Data were analysed using the Geneious ClustalW. Oligonucleotide primer sequences are listed in Table S5.

DNA from frozen pellets was isolated using the DNeasy Blood and Tissue kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. DNA was quantified with a Qubit fluorometer (Thermo Scientific, Raleigh, NC) according to the manufacturer’s instructions and normalized to 10 ng/μl. Samples with (DNA) less than 10 ng/μl were not diluted. Twenty microliters of the normalized (or undiluted, in the case of low concentration samples) DNA was used as template in a PCR using primers flanking the transposon barcode region, each containing an Illumina adapter and multiplexing index sequence (BarSeq) [23 ,24 (link)]. PCR was performed using Q5 DNA polymerase with Q5 GC enhancer (New England Biolabs, Ipswich, MA) for 25 cycles of 30 seconds at 98°C, 30 seconds at 55°C, and 30 seconds at 72°C, followed by a final extension at 72°C for 5 minutes. Following PCR, 10 μl of each reaction was pooled into 3 sets of 25 (sets A, B, and C; see S1 Data) amplicon libraries, corresponding to each experimental set (see previous section). Three pooled libraries (representing sets A, B, and C) were then purified using the DNA Clean & Concentrate Kit (Zymo, Irvine, CA) according to the manufacturer’s instructions. Each set was sequenced on its own lane on an Illumina HiSeq 2500 machine using the 1T paired-end, 2 x 101 cycle protocol, producing an average of 3 to 8 million reads per sample.