Dna clean and concentrator 5

The semidwarfing gene d60 was transferred into Koshihikari by consecutive backcrosses to prepare a semidwarf Koshihikari named Koshihikari d60Gal line. Whole-genome analysis was conducted using the Koshihikari d60Gal line and Koshihikari (D60gal). Genomic DNAs were extracted from each cultivar using the hexadecyltrimethylammonium bromide (CTAB) method. Genomic DNA was tagged and fragmented to average 500-bp long using Nextera® transposome. After purification of the transposome using DNA Clean and Concentrator^TM-5 (Zymo Research, Irvine, CA, USA), adaptors for fixation on the flow cell were synthesized at both ends of each fragment using polymerase chain reaction (PCR). Then, the DNA fragments were subjected to size selection using AMPure XP magnetic beads (Beckman Coulter, Brea, CA, USA). Finally, qualitative and quantitative measurements using a Fragment Analyzer^TM (Advanced Analytical Technologies) and Qubit^® 2.0 Fluorometer (Life Technologies; Thermo Fisher Scientific, Inc., Waltham, MA, USA) were performed to prepare a DNA library for NGS. The resulting sequenced reads were mapped with BWA software using the Nipponbare genome as a reference, followed by the detection of Single Nucleotide Polymorphisms (SNPs) and Indels using SamTools software.

Multiple-locus variable nucleotide tandem repeat (MLVA) analysis was performed for 16 of the tdh+, trh+ V. parahaemolyticus strains, employing nine primer sets belonging to both chromosomes 1 and 2. PCR conditions were identical to those described for conventional PCR. After confirmation by PCR, 25 μl PCR product was purified using DNA Clean and Concentrator^TM-5 (ZymoResearch, Irvine, CA, United States) and mailed for sequencing (Eurofins MWG Operon, Louisville, KY, United States). After sequencing, the number of repeat motifs were counted for each isolate at each individual loci. Primers and repeat motifs for each loci can be found in Table 2.

All sequence-verified plasmids were injected to either y¹ w* P{nos-phiC31}; P{CaryP}attP40 (#79604) or y¹ w* P{nos-phiC31}; P{CaryP}attP2 for integration into the second or third chromosome, respectively. To verify the sequences of the newly generated transgenic lines, 232 bp PCR products were amplified from genomic DNA using hsp70p_F and shRNA_GA_R1 primers, purified using DNA Clean and Concentrator-5 (Zymo Research), then sequenced using shRNA_GA_R1 primer.

Plasmid DNA (pUC19, NEB) (50 ng μL^–1), an AdoMet cofactor analogue (150 μM Ado-6-amine or Ado-6-azide), and M.TaqI DNA methyltransferase (0.1 mg ml^–1) were incubated in CutSmart buffer (New England Biolabs) for 2 h at 60 °C. Subsequently, 1 μL of proteinase K was added and the reaction was incubated for 1 h at 55 °C. The product was purified using silica-based columns (DNA Clean and Concentrator-5, Zymo Research) and eluted with 25 μL of Milli-Q water.

Samples containing at least 250 ng DNA were selected for DNA-methylation profiling (n = 23). DNA bisulfite conversion was undertaken using the ZymoEZ DNA methylation kit (Zymo research, USA), then treated with FFPE DNA Restore kit (Illumina, San Diego, CA, USA) and DNA clean and concentrator-5 (Zymo research, USA). Standard quality controls confirmed DNA quantity/quality and bisulfite conversion. The DNA was then processed using the Illumina Infinium HumanMethylation EPIC Bead-Chip array (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions. The iScan control software was used to generate raw data files from the BeadChip in .idat format, analysed using GenomeStudio version 2.0 (Illumina, San Diego, CA, USA) and were checked for quality measures according to the manufacturer’s instructions.

The SPAAC reaction was performed in a variety of different solvents and solvent mixtures (water, ethanol, DMF and DMSO (Sigma-Aldrich)). 1 μg of azide coupled pUC19 DNA was incubated with 50 μl of the cyclooctyne–rhodamine dye at a concentration of 1 mM. The reaction mixtures were incubated overnight at room temperature and then purified using a silica-based column (DNA Clean and Concentrator-5, Zymo Research) and eluted with water.