Genejet plant genomic dna purification kit

Prior to DNA extraction, the colonial mycelium was sub-cultured in potato dextrose broth (PDB) to get a larger yield of DNA. Subsequently, liquid nitrogen, and a pestle and mortar were used to grind the tissue into powder. Then 150–200 mg of ground tissue was added to a 2 ml Eppendorf tube for DNA extraction, the process of which followed the protocol provided by Thermo scientific GeneJET Plant Genomic DNA Purification Kit. Finally, the DNA was stored at −20 °C for future use (Saleh et al., 2017 ).

The copy number of the integrated KO constructs was determined using a qPCR-based method^{78 (link)}. Genomic DNA was extracted from 100 mg frozen protonema using the GeneJET Plant Genomic DNA Purification Kit (Thermo Scientific, Waltham, USA). DNA concentrations were adjusted to 3 ng/µL for each sample and qPCR was performed with primers for the 5′ and 3′ flanks as well as with primers for the corresponding selection cassette. Additionally, primers for the single copy gene CLF (Pp3c22_2940V3) were used as internal control. Reference lines were WT as well as in-house lines with known single integrations of the used selection cassettes. Primers are listed in Supplementary Data S5. PCR reactions were done using 2x SensiFAST Mix (Bioline, London, UK) and analyzed in a Lightcycler 480 II (Roche, Basel, Schweiz).

Brassica rapa ssp trilocularis variety R-o-18 were grown in a greenhouse at 70°/60°F (day/night) under at least 16 h of illumination. Plants were fully dried before seed collection. Dry seeds were soaked in water for no more than 60 min before manual dissection to remove mature embryos. Three wild-type or five rdr2 mutant embryos were pooled before DNA extraction with the GeneJET Plant Genomic DNA Purification Kit (Thermo Fisher Scientific, K0791). Embryos from rdr2/RDR2 heterozygous mothers were individually collected, prepped, and genotyped prior to DNA pooling. rdr2 mutants were used in this study due to their complete loss of 24-nt siRNAs and strong developmental phenotype. Torpedo-stage endosperm and embryo samples were dissected from pistils that were manually pollinated with B. rapa genotype R500. Whole-genome bisulfite sequencing libraries were prepared as previously described [44 (link)]. Lambda Phage DNA (Promega D1521) was included as a bisulfite conversion control. Libraries were pooled and sequenced in a single lane of paired-end 76 nt on an Illumina NextSeq500 at the University of Arizona Genetics Core.

The most potent fungal isolate in tannase production in this study was isolated from tea waste. The selected isolate was identified genetically using 18S rRNA-based molecular technique. DNA extraction was done using protocol of Gene Jet Plant Genomic DNA Purification Kit (Thermo-Scientific, K0791, made in Germany). Primers used for PCR and DNA sequencing are ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCT TATTGA TATGC-3′). The PCR conditions were run as follows: initial denaturation at 96 °C for 3 min followed by 25 cycles of denaturation at 96 °C for 30 s, annealing at 55 °C for 30 s and an extension step at 72 °C for 1 min. The purified PCR product was sequenced, and these sequences were subjected to BLAST similarity analysis available from NCBI Gen Bank database. A phylogenetic tree was constructed using the Tamura-neighbor joining method by MEGA X software (Molecular Evolutionary Genetics Analysis, Bioinformatics, Tokyo Metropolitan University, Hachioji, Tokyo, Japan).

Genomic DNA of D. sukatschewii and D. cespitosa were isolated from young leaves of the studied accessions using the GeneJet Plant Genomic DNA Purification Kit (Thermo Fisher Scientific, Vilnius, Lithuania). The quality of the DNA samples was checked with the Implen Nano Photometer N50 (Implen, Munich, Germany). The concentration and purification of the extracted DNAs were assessed with the Qubit 4.0 fluorometer and Qubit 1X dsDNA HS Assay Kit (Thermo Fisher Scientific, Eugene, OR, USA).
For D. sukatschewii and D. cespitosa, whole genome sequencing with low coverage was performed at the Beijing Genomics Institute (BGISeq platform) (Shenzhen, Guangdong, China) according to the NGS protocol for generating 5–10 million of paired-end reads of 150 bp in length, which provided at least 0.147–0.350× of the coverage of the D. cespitosa genome (1C = 4283.64–5105.16 Mbp, Eurasian region) [36 (link),37 (link)]. The raw sequencing data for D. cespitosa (SAMN26938767) and D. sukatschewii (SAMN26938768) were uploaded to the National Center for Biotechnology Information (NCBI) BioProject database under accession number PRJNA819861 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA819861, accessed on 25 March 2022).