Wizard magnetic dna purification system for food

To prepare samples containing the exogenous and endogenous target sequences, genuine seeds from the GM rice varieties TT51-1, Kangyou97, Kefeng6, Kefeng8, KMD1, Bar68-1, and Bar66-1 as well as nontransgenic rice seeds were collected by members of our laboratory. Plant genomic DNA was isolated from rice flour using the Wizard Magnetic DNA Purification System for Food according to the manufacturer's instructions (Promega, Madison, WI, USA). The DNA pellets were dissolved in 100 μL of nuclease-free water. DNA quantification was performed using a PicoGreen assay (Quant-iT PicoGreen dsDNA Kit, Invitrogen, Carlsbad, CA, USA), and the samples were diluted to a 50 ng/μL working stock, which was stored in aliquots at −80°C. Restriction enzymes were purchased from New England Biolabs (Ipswich, MA). DNA synthesis, primer synthesis, and DNA sequencing were performed by Genewiz, Inc. (Suzhou, China).

Genomic DNA from pure cultures of bacterial strains was extracted using a DNeasy blood and tissue kit (Qiagen, SA, Courtaboeuf, France), according to the manufacturer’s instructions^{59 (link)}. Total DNA extraction from breads was carried out using the Wizard Magnetic DNA Purification System for Food (Promega, Madison WI), following the manufacturer’s instructions. Each DNA extraction was performed in triplicate.

Fungal DNA from compost was extracted using a modified version of Sturrock et al. (2015) (link). Compost from two biological replicate plants were combined into one sample (2 g) for each treatment of each variety. For homogenization, each sample was placed in a 50 ml falcon tube with three 1/4 inch ceramic balls (MP Biomedicals, United States), 15 ml of CTAB buffer (cetyltrimethylammonium bromide) and 0.45 ml of Antifoam B and homogenized using a FastPrep-24^TM homogeniser (MP Biomedicals, United States). DNA extraction was performed using the Wizard Magnetic DNA Purification System for Food as per manufacturer’s instructions (Promega Wizard Food Kit, Southampton, United Kingdom).

One hundred and thirty-four Alternaria mono-conidial strains were cultured for three days on cellophane disks overlaid on PDA plates at 25 °C. Afterwards, mycelium of each strain was collected by scraping, frozen, and lyophilized.
Genomic DNA was extracted and purified from powdered lyophilized mycelia (10–15 mg) using the “Wizard Magnetic DNA Purification System for Food” kit (Promega Corporation, Madison, WI, USA), according to the manufacturer’s protocol. Quantity and integrity of DNA were checked at Thermo-Scientific Nanodrop (LabX, Midland, ON, Canada) and by comparison with a standard DNA (1 kb DNA Ladder, Fermentas GmbH, St. Leon-Rot, Germany) on 0.8% agarose gel after electrophoretic separation.

The antagonistic strain T. atrobrunneum (formerly T. harzianum) ITEM 908 was originally isolated from soil collected in Apulia, Italy, and maintained in the Agri-Food Toxigenic Fungi Culture Collection of the Institute of Sciences of Food Production, CNR, Bari (http://www.ispa.cnr.it/Collection). A monoconidial culture was grown for 5 days on PDA (Potato Dextrose agar, Oxoid, Italy). Fungal mycelium was then scraped off the agar surface and ground in a mortar using liquid nitrogen to a fine powder. DNA extraction was performed from 10 mg of lyophilized material using the “Wizard ® Magnetic DNA purification system for Food” (Promega, Madison, WI, USA), according to the manufacturer’s instructions. The quantity and quality of isolated DNA was determined a NanoDrop-2000 (Thermo Fisher Scientific, Wilmington, DE, USA) and a Qubit 3.0 fluorometer (Life Technologies). DNA was then subjected to whole genome shotgun sequencing using the Ion S5™ library preparation workflow (Thermo Fisher Scientific, Waltman, MA, USA). Four hundred bp mate-paired reads were generated on the Ion S5™ System (Thermo Fisher Scientific). Duplicate reads were removed by FilterDuplicates (v5.0.0.0) Ionplugin. De novo assembly was performed by AssemblerSpades (v.5.0) Ionplugin™.

The EBN samples were extracted using commercial kit, Wizard^® Magnetic DNA purification system for food (Promega Corporation, Madison, WI, USA) following the manufacturer’s instructions except the volume adjustments in lysis buffers. Each EBN of 25 mg was vigorously vortexed with 450 µL of Lysis Buffer A and 5 µL of RNase A, then vortexed again with 200 µL of Lysis Buffer B for 15 s in a 1.5 mL microcentrifuge tube. The tube was laid on its side and incubated at room temperature for 10 min. The sample was vigorously vortexed with 700 µL of precipitation solution and centrifuged at 13,000× g for 10 min in a 5415D microcentrifuge (Eppendorf, Hamburg, Germany) for protein precipitation. About 700 µL of supernatant was vortexed with 50 µL of resuspended MagneSil™ paramagnetic particles (PMP) in a new microcentrifuge tube, and then continued with remaining procedures in the manufacturer’s instructions. The Wizard method lyses with guanidine thiocyanate and RNase, and binds DNA to silica-coated magnetic beads.