Genelute plant genomic dna miniprep kit

After 48 h incubation under total darkness, the protoplast cells were washed with W5 solution for several times in order to remove non-transfected vectors in the culture. Total genomic DNA was isolated from the incubated protoplast and control (without pCAMBIA1303 transfection) according to manufacturer instructions (GenElute^TM Plant genomic DNA miniprep kit; Sigma-Aldrich, Burlington, VT, USA).The presence of hptII and gusA in the transfected protoplasts was confirmed by a PCR analysis using gene-specific primers, the HiPi Master Premix (Elpis Biotech, Daejeon, Korea), and the following program: 94 °C for 5 min; 30 cycles of 94 °C for 1 min, 55 °C for 1 min, and 72 °C for 1 min; 72 °C for 7 min. The forward and reverse primers for amplifying gusA (515 bp) were, respectively, FP (5′-ATTGATCAGCGTTGGTGG-3′) and RP (5′-ACGCGTGGTTACAGTCTTGC-3′), whereas the forward and reverse primers for amplifying hptII (407 bp) were, respectively, FP (5′-GATGTTGGCGACCTCGTATT-3′) and RP (5′-GTGTCACGTTGCAAGACCTG-3′). The PCR products were analyzed by 1% (w/v) agarose gel electrophoresis and ethidium bromide staining.

Seeds of each rice genotype were germinated in a growth chamber with 10 hours light, 85% relative humidity and 30°C temperature. At the three-leaf stage, leaf tissues from five seedlings of each genotype were collected for DNA extraction. The total genomic DNA was then isolated from this leaf tissue using GenElute^TMPlant Genomic DNA mini prep kit (Sigma, USA) following the manufacturer’s protocol.

Total genomic DNA extractions were performed on the177 accessions. DNA isolation was performed using the GenEluteTM Plant Genomic DNA miniprep kit (Sigma, St. Louis, MO). The quality of the DNA was analyzed by ND-1000 Spectrophotometer (Nanodrop Technologies, Wilmington, DE) and by electrophoresis on agarose gel. The concentration of DNA was estimated using Qubit® 2.0 Fluorometer (Life Technologies, Singapore) and Qubit® dsDNA BR Assay Kit (Life Technologies, Eugene, OR).

For the GBS of the melon collection, leaf tissue was taken from 44 of the 51 accessions, listed in Table 1. Total genomic DNA isolation was performed as described by Gur et al. [37 (link)] using the GenEluteTM Plant Genomic DNA miniprep kit (Sigma, St. Louis, MO, USA). The quality of the DNA was analyzed with an ND-1000 spectrophotometer (Nanodrop Technologies, Wilmington, DE) and by electrophoresis on agarose gel. The concentration of DNA was estimated using a Qubit^® 2.0 Fluorometer (Life Technologies, City, Singapore) and a Qubit^® dsDNA BR Assay Kit (Life Technologies, Eugene, OR, USA).

DNA extraction, PCR amplification and sequencing for Aspergillus spp. were done in the present study. Conidia of the Aspergillus isolates used for the molecular studies were grown on 0.5 mL malt peptone (MP) broth in a 1.5 mL Eppendorf tube. The cultures were incubated at 25 °C for 3–7 d. Grown mycelium was scraped off and about 100 mg mycelium from each isolate was used for genomic DNA extraction. The mycelia were ground into a fine powder using tissue-grinding pestles. The powder was transferred into a 1.5 mL sterile Eppendorf tube and stored at −20 °C until analysis. DNA was extracted from the cells using GenElute^TM Plant Genomic DNA Miniprep Kit (purchased from Sigma Aldrich, Darmstadt, Germany) according to the recommendations of the manufacturer. The highly genomic DNA was stored at −20 °C until analysis.

A set of 99 Kuwaiti Rhanterium landraces were used in this study. They were collected from the areas of Al Kabd (n = 21), Sabah Al Ahmed Nature Reserve (SANR, n = 18), Om Qaser (n = 8), Al Maqwa (n = 8), Al Salmi (n = 24) and Mina Abdulla (n = 19). The GPS coordinates of each specimen were recorded and used to mark the locations on Google Maps (Figure 6). The GPS coordinates and soil types of these populations are provided in Table S3. The average annual precipitation ranges between 75 and 160 mm/yr. The temperature in Kuwait falls between 0 and 46 °C. Genomic DNA was extracted using a GenElute^TM Plant Genomic DNA Miniprep Kit (Sigma, St. Louis, MO, USA), as per the manufacturer’s instructions, from young leaves [15 ]. All the DNA samples were quantified through a Qubit fluorometer (Thermo Fisher Scientific, Carlsbad, CA, USA) employing the Qubit ^®BR dsDNA Assay (Life Technologies, Inc., Grand Island, NY, USA).

Genomic DNA was extracted from 100 mg of leaves of each line and of the set of standard cultivars using a GenElute^TM Plant Genomic DNA Miniprep kit (Sigma-Aldrich), following the manufacturer’s protocol. A list of the primer pairs used to assay Glu-A1 and Glu-B1 and the relevant PCR conditions are given in Suppl. Table 2. The 10 µL PCRs were based on GoTaqHotStart® colorless Mastermix 2X (Promega, Madison, WI, USA) and the reaction conditions replicated those given by^{35 (link)}. The amplicons were electrophoretically separated through Tris acetate EDTA agarose gels.