Purelink genomic plant dna purification kit

The test material used for apple genetic transformation was the tissue culture seedlings of clone “GL-3”, with strong regenerative ability and high transformation efficiency, obtained from the “Gala” apple. The leaves of “GL-3” were transformed with R. rhizogenes strain K599, which carries the sequence MdUGT73CG22-PBI121, and 50 mg·L⁻¹ kanamycin and 250 mg·L⁻¹ sodium cefotaxime were added to the MS medium for the selection of regenerating shoots. During the selection process, slow-growing and yellowing regeneration shoots were discarded, and normal-growing regeneration shoots continued to be cultured on the selection medium until they developed into robust individuals with stem and leaf tissues.
DNA was extracted from “GL-3” and transgenic apple plants using the PureLink™ Genomic Plant DNA Purification Kit (K183001, Thermo Fisher Scientific, Waltham, MA, USA). The partial sequence of the CaMV 35S promoter on the vector was used as the upstream primer, the downstream primer was designed with the sequence of MdUGT73CG22 gene for PCR amplification, and electrophoresis was carried out to detect whether it was the target gene or not.

DNA was isolated by collecting six young leaves (100 mg) from each accession. Two leaves were considered for each accession/replicate. Once harvested, the tissue was lyophilized in microcentrifuge 2-ml tubes and then disrupted to a fine powder using a Tissue Lyser II (Qiagen, Hilden, Germany) at 30 strokes per second for 30 s. Nucleic acid isolation was then performed using the PureLink™ Genomic Plant DNA Purification Kit (Thermo Fisher Scientific, Waltham, MA, USA). DNA concentration and quality were measured by absorbance at 260 and 280 nm, respectively, using both UV–Vis spectrophotometer (ND-1000; NanoDrop, Thermo Scientific, Wilmington, DE, USA) and Qubit 2.0 Fluorometer based on Qubit dsDNA HS Assay (Thermo Fisher Scientific, Waltham, MA, USA). The DNA solution was then diluted to a working concentration with distilled water. Genotyping was performed using the Illumina 10K SolCAP Tomato Infinium array (Illumina Inc., San Diego, CA, USA) yielding a total of 7,720 SNPs (Sim et al., 2012a (link)). SNP calls, through the translation of the signals of fluorescent dyes into genotype groups, were obtained using GenomeStudio version 1.7.4 (Illumina Inc., San Diego, CA, USA).

Initial verification of transgenic status was performed on in vitro leaves. Leaves were harvested from putative transgenic plants and non-transgenic control and verified by PCR utilizing the Extract-N-Amp™ Plant PCR Kit (Sigma-Aldrich, St. Louis, MO) and gene-specific primers [42 ]. Total RNA was isolated utilizing the RNeasy Mini Kit (Qiagen Inc. Valencia, CA) according to the manufacturer’s protocol. A 1-μg aliquot of RNA was reverse transcribed using RevertAid First-Strand cDNA Synthesis Kit (Thermo Fisher Scientific Inc). Real-time quantitative PCR (qPCR) was performed using the StepOne Plus system (Thermo Fisher Scientific Inc) utilizing SYBR Green reagents. The relative mRNA levels were compared to those of the Citrus sinensis glyceraldehyde-3-phosphate dehydrogenase (GAPC) gene [43 (link)] and calculated using the 2^-ΔΔCT method [44 ]. Citrus genomic DNA from one-year-old transgenic trees and non-transgenic control was isolated using the PureLink™ Genomic Plant DNA Purification Kit (Thermo Fisher Scientific Inc). Transgene presence was re-confirmed by PCR. The transgene copy number in the selected lines was also validated using qPCR, essentially as previously described [45 , 46 ]. All primer sequences used in this study are listed in Table 1.

Total genomic DNA was isolated using the PureLink™ Genomic Plant DNA Purification Kit (part #, K183001, Thermo Fisher Scientific Inc., Waltham, MA, USA), according to the manufacturer’s instructions. Briefly, 30 mg of the sample was weighed and transferred into a 1.5 ml centrifuge tube. Lysis buffer and Proteinase K from the kit were added, followed by vortexing for 1 min and pulse spinning. The tube was incubated at 56 °C for 1 h with occasional swirling. The other steps were the same as outlined in the previously published protocol. The obtained DNA concentration was quantified on a NanoDrop 2000 spectrophotometer and Qubit 3.0 (Thermo Fisher Scientific).

Based on the gene expression and bioassays, the best performing transgenic cotton line (NIBGE-E2) was selected and further used for event characterization using NGS technology. Highly purified good quality genomic DNA was isolated from confirmed -triple gene NIBGE-E2 cotton plant by using PureLink™ genomic plant DNA purification kit (Catalog No. K183001, Thermo Fisher Scientific) and single replicate of NIBGE-E2 cotton was sequenced on BGISEQ-500 platform. Sequencing data in the form of paired-end reads stored in FASTQ format was obtained and data filtering was done to remove adapters, noisy or low-quality base ratio reads, and reads with unknown 'N' bases. The cleaned data were aligned to the reference cotton genome using Burrows-Wheeler Aligner (BWA) (Li 2013); followed by alignment with the triple gene NIBGE cotton construct sequence. The alignment was performed in CLC Genomics Workbench to detect the event in the genome of NIBGE triple gene cotton (. For NGS-based detected events in triple gene cotton, the event-specific primers (Supplementary Table 2) were designed from the junction region of the cotton genome and triple gene construct to validate the results. Event-specific primers were thoroughly evaluated and confirmed to be different from Monsanto cotton BGI (Mon531) and BGII (Mon15985) events.