Plant genomic dna extraction kit

Using the same plant material, the cut ends of the leaves were collected every day for 9 days after clipping and daubing with water or BSA. Unclipped leaves were used as controls. Each sample for DNA extraction consisted of ten 1-cm-long leaf pieces. The leaves were flash-frozen in liquid nitrogen and stored at −80°C. The leaf tissues were ground to a fine powder in liquid nitrogen using a mortar and pestle, and the DNA was extracted using a plant genomic-DNA extraction kit (TIANGEN, Beijing, China) according to the manufacturer’s protocol. The total DNA was treated with RNase A (TaKaRa Bio Inc., Dalian, China) to remove any contaminating RNA. The isolated DNA, which was mainly derived from apoptotic cell bodies, was electrophoresed on a 2.0% agarose gel at 50 V for 3 h. The DNA fragments, which consisted of 160–200 bp multimers, were visualized under ultraviolet light after staining with ethidium bromide.

When the plants were at a height of approximately 20 cm, 1 mL L^-1 10% Basta solution (8.0 mg l^-1) was used for preliminary screening. The plants were sprayed three times every 6 days. Genomic DNA was extracted from the alfalfa leaves using a Plant Genomic DNA extraction kit (Tiangen, Beijing). The PCR was conducted with a genomic DNA template in PCR premix using two convergent primers that were complementary to the CsALDH gene and another pair of primers that were complementary to the bar gene. DNA amplification was performed at 94°C for 3 min; 35 cycles of 94°C for 30 s, 50°C for 45 s, and 72°C for 1 min 30 s, and then a final extension at 72°C for 10 min. Total RNA was extracted using the UNIQ-10 column total RNA extraction kit (Sangon Biotech, Shanghai). The RT-PCR conditions were identical for both CsALDH and MsActin, as described in the genomic PCR analysis. The PCR products were separated on a 1.2% agarose gel, stained with GelStrain (TransGene Biotech, Beijing), and visualized under UV. Sixteen transgenic plants showing highly expressed CsALDH were selected for further stress tolerance and phenotype analysis. All subsequent experiments were conducted on cloned plants from the T₀ generation of transgenic plants.

Since the G. lindleyana plant does not have typical leaf organs, its root was used to obtain its chloroplast genome sequence. The root of G. lindleyana (Figure 1) was collected from Yuexi in Anhui Province, China (30.84°N; 116.34°E) at an altitude of 1130 m on October 2, 2020. This plant was identified morphologically, and its voucher specimen deposited in the Institute of Medicinal Plant Development, Chinese Academy of Medicinal Sciences.
Fresh root samples were ground into fine powder with liquid nitrogen in a mortar, and then used to extract the genomic DNA using a Plant Genomic DNA Extraction Kit (Tiangen Biotech (Beijing) Co., Ltd., China). The DNA concentration and the ratios of A260/A280 and A260/A230 were measured using a Thermo Scientific NanoDrop 2000 ultra-micro spectrophotometer (Thermo Fisher Scientific Inc., MA, USA). Following the construction of a 270 bp PCR-free library, the whole genomic DNA of G. lindleyana was sequenced using the Illumina NovaSeq 6000 platform via shotgun sequencing.

DNA was extracted by the Plant Genomic DNA Extraction Kit (DP305, TIANGEN, Beijing, China). Total RNA was extracted by the RNAprep Pure Plant Kit (Polysaccharides & Polyphenolics-rich) (DP441, TIANGEN, Beijing, China). The remaining DNA from the extracted RNA was digested by RNase-free DNase I (EN0521, Thermo, Waltham, MA, USA). Then, the quality and concentration of DNA and RNA were examined by Nano Drop 2000 (Thermo, Waltham, MA, USA).

DNA was isolated from the 2-month-old castor bean plants of each of the 200 RILs. DNA extraction was carried out using the plant genomic DNA extraction Kit (TIANGEN, Beijing, China) following the manufacturer’s instructions. RNase A was then added to digest RNA. The quality and concentrations of DNA were detected using a NanoDrop 2000 (Thermo Fisher Scientific, USA), while DNA integrity was examined by electrophoresis on 1% agarose gels.
To prepare the reduced representation libraries for sequencing, the GBS protocol was carried out according to the method reported by Elshire et al.^{17 (link)}. In brief, the genomic DNA was first digested by restriction enzymes. In this case, EcoRI and MseI were selected to efficiently reduce genome complexity. The barcode adapters and common adapters were then linked to the sequence ends of the fragmented DNA samples. PCR amplification and fragment selection was performed. Libraries were then sequenced by Illumina HiSeq3000 platform, which generated 150 bp paired-end reads.

Plant materials in this study include the L. indica var. Black Diamond ‘Blush V2’ and four genotypes of Arabidopsis, Col-0 (WT), atsos2 mutant (T-DNA SALK_056101C), LiCIPK30/atsos2 (COM) and LiCIPK30/WT (OE). All Arabidopsis lines were germinated on half-strength Murashige & Skoog media supplied with 2% sucrose (1/2 MS) for 10–14 days, then the seedlings were planted on mixed soil (50% Pindstrup Substrate and 50% vermiculite) and grown in an artificial climate chamber (16 h day/8 h night, 22°C day/18°C night).
The chemical reagents used in this study were purchased from SINOPHARM (Beijing, China) or Ameresco (Framingham, MA, United States). The RNA extraction reagent (MiniBEST Plant RNA Extraction Kit), first strand cDNA synthesis kit (PrimeScript™ RT reagent Kit with gDNA Eraser), and TB GREEN reagents for quantitative polymerase chain reaction (qPCR) were purchased from TaKaRa (Beijing, China). The plant genomic DNA extraction kit was purchased from TIANGEN (DP305, Beijing, China), and plasmid DNA extraction kit from Beyotime (Shanghai, China). Primers were synthesized by General Bio (China, Anhui, Chuzhou). All oligo primers used in this study are listed in Supplementary Table 1.