The genomic DNA extraction from root-associated bacterial endophytes was performed using the DNAiso Reagent Kit (TaKaRa Biotechnology, Co., Ltd, Dalian, China) according to the manufacturer’s instructions. The quantity and quality of genomic DNA were observed by the Nano Drop 2000c Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) at the 260 nm and 280 nm. The purity of the extracted bacterial genomic DNA samples was further assessed through the 1% agarose gel electrophoresis with a 1 x TAX buffer solution, following the described procedure of Koh et al. [71 (link)].
Dnaiso reagent kit
Manufactured by Takara Bio
Sourced in China
The DNAiso Reagent Kit is a reagent kit designed for the isolation and purification of DNA from various biological samples. The kit utilizes a guanidinium thiocyanate-based extraction method to effectively remove contaminants and obtain high-quality DNA.
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Lab products found in correlation
Nanodrop 2000c spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Cellomics arrayscan vti hcs reader, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Sybr fast qpcr mix, by Takara Bio (1 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (1 mentions)
Minibest dna fragment purification kit, by Takara Bio (1 mentions)
5 protocols using dnaiso reagent kit
1
Bacterial Genomic DNA Extraction
2
Mitochondrial Mass and mtDNA Quantification
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The mitochondrial mass was measured using the fluorescent dye NAO (Thermo Fisher Scientific Inc., Carlsbad, CA, USA). NAO was added to SH-SY5Y cells to achieve a final concentration of 10 μM for 10 min at 37 °C after the cells were treated. The fluorescence intensity was detected and analysed with a Cellomics ArrayScan VTI HCS Reader (Cellomics Inc., Pittsburgh, PA, USA) with the Morphology Explorer BioApplication software. The mitochondrial mass was quantified by the value of the average fluorescent intensity of NAO.
Quantification of mtDNA was carried out using real-time qPCR. Total DNA was extracted using a DNAiso Reagent Kit (TaKaRa, Liaoning, China), followed by qPCR using SYBR® Fast qPCR Mix (TaKaRa, Liaoning, China). The relative mtDNA copy number was calculated as the ratio of mtDNA/nDNA. The primer sequences are listed in Supplementary TableS2 .
For reverse transcription (RT)-qPCR, total RNA was extracted using the TRIzol reagent (Life Technologies, Rockville, MD, USA). RT-qPCR was performed using the PrimeScript™ RT Reagent Kit (Perfect Real Time) and SYBR® Fast qPCR Mix in accordance with the manual guide. β-ACTIN mRNA was used as the internal control. The primer sequences are listed in Supplementary TableS2 .
Quantification of mtDNA was carried out using real-time qPCR. Total DNA was extracted using a DNAiso Reagent Kit (TaKaRa, Liaoning, China), followed by qPCR using SYBR® Fast qPCR Mix (TaKaRa, Liaoning, China). The relative mtDNA copy number was calculated as the ratio of mtDNA/nDNA. The primer sequences are listed in Supplementary Table
For reverse transcription (RT)-qPCR, total RNA was extracted using the TRIzol reagent (Life Technologies, Rockville, MD, USA). RT-qPCR was performed using the PrimeScript™ RT Reagent Kit (Perfect Real Time) and SYBR® Fast qPCR Mix in accordance with the manual guide. β-ACTIN mRNA was used as the internal control. The primer sequences are listed in Supplementary Table
3
Isolation and Genome Sequencing of Sporidiobolus pararoseus
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S. pararoseus NGR was isolated from strawberry fruit in the greenhouse of Shenyang Agricultural University (41°49′N, 123°34′E) in Shenyang City, Liaoning Province, China. Species identification was performed through morphological and molecular methods. The available GenBank accession number of S. pararoseus NGR 26S rDNA is HM749332. The strain number is recorded in the China General Microbiological Culture Collection Center as CGMCC 2.5280. S. pararoseus NGR cultures were grown for 72 h in 250 mL Erlenmeyer baffle flasks containing 50 mL of the YPD medium (10 g/L yeast extract, 20 g/L peptone and 20 g/L glucose, pH 6.5 ± 0.5) at 28 °C on a rotary shaker at 180 rpm. Genomic DNA of S. pararoseus NGR was extracted using the DNAiso Reagent kit (Code No.: 9770A) (Takara Bio, Dalian, China) according to the manufacturer’s protocols. The extracted genomic DNA was subjected to quality control by agarose gel electrophoresis and quantified by Qubit 2.0 fluorometer (Life Technologies, USA). The obtained genomic DNA (≥500 ng/μL) was used for whole genome sequencing and PCR verification.
4
Dairy Goat Sperm Sexing Protocol
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Ten fresh semen samples (six replicates for each sample) were diluted to 600 × 104/mL using the pH 6.8 diluents, and it was centrifuged at 1,000 × g for 5 min. The supernatant was discarded and added slowly with 10 mL different pH diluent. Then, the tubes were tilted to 45° and incubation at 37°C with 5% CO2. Finally, 10 μL upper sperm was taken at 10 min intervals, and genomic DNA was extracted using a DNAiso Reagent kit (9770A; TaKaRa).
To calculate the X/Y sperm ratios in isolated upper sperm and analyze the X and Y sperm bioinformatics of dairy goats, primers were designed by selecting the F9 gene specific to the X chromosome and the ZFY gene specific to the Y chromosome (Table 1 ). The genomic DNA of dairy goat sperm was used as a template for PCR amplification. The PCR product was ligated to T-Vector pMDTM 19 and transferred to E. coli DH5α competent cells by the heat shock method to prepare a positive standard. By optimizing the PCR reaction system and reaction conditions, we established a standard curve and verified the specificity, sensitivity, and stability of the method. Subsequently, the double TaqMan qPCR (Thermal Cycler CFX96 Real-Time System, United States) method was employed to calculate the X/Y sperm ratio.
To calculate the X/Y sperm ratios in isolated upper sperm and analyze the X and Y sperm bioinformatics of dairy goats, primers were designed by selecting the F9 gene specific to the X chromosome and the ZFY gene specific to the Y chromosome (
5
Muscle Tissue DNA Extraction Protocol
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Total DNA from muscle tissues was extracted using a DNAiso reagent kit (TaKaRa), treated with ribonuclease A and purified with the MiniBEST DNA fragment purification kit (TaKaRa). No RNA contamination was detected upon agarose gel electrophoresis, and DNA concentration was measured using a nucleic acid/protein analyser (ND-1000; NanoDrop).
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