EasyPure Genomic DNA kit and TransScript Green Two-Step qRT-PCR SuperMix (EE101-01 and AQ201-01, respectively; both from TransGen Biotech Co., Ltd.) were used.
Easypure genomic dna kit
Manufactured by Transgene
Sourced in China
The EasyPure Genomic DNA Kit is a laboratory equipment designed for the rapid and efficient extraction of high-quality genomic DNA from a variety of biological samples. The kit utilizes a simple and streamlined protocol to isolate DNA, making it suitable for a range of applications in molecular biology and genetics research.
Automatically generated - may contain errors
Lab products found in correlation
Ez dna methylation gold kit, by Zymo Research (5 mentions)
Rq1 rnase free dnase, by Promega (3 mentions)
Pmd19 t, by Takara Bio (3 mentions)
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Easytaq pcr supermix, by Transgene (3 mentions)
Nanodrop 2000 uv spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Easypure quick gel extraction kit, by Transgene (2 mentions)
Hiseq 2000, by Illumina (2 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (2 mentions)
Easypfu dna polymerase, by Transgene (2 mentions)
Relaxgene blood dna system, by Tiangen Biotech (2 mentions)
Qubit 2, by Thermo Fisher Scientific (2 mentions)
Qiaamp circulating nucleic acid kit, by Qiagen (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Transscript green two step qrt pcr supermix, by Transgene (1 mentions)
Trizol plus purification kit, by Thermo Fisher Scientific (1 mentions)
Primescript first strand cdna synthesis kit, by Takara Bio (1 mentions)
Trizol extraction reagent, by Thermo Fisher Scientific (1 mentions)
99 protocols using easypure genomic dna kit
1
Efficient DNA Extraction and qRT-PCR
2
Butterfly DNA Extraction and Sequencing
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Genomic DNA was extracted from 95–100% ethanol-preserved muscle tissue of two adult butterfly legs, using an EasyPure Genomic DNA Kit according to the manufacturer’s instructions (TransGen Biotech Co., Led., Beijing, China). Extracted genomic DNA was eventually dissolved in 80 µL ddH2O and kept in a freezer (–20 °C) until it was used for polymerase chain reaction (PCR). Sequences of six nuclear and mitochondrial genes (EF-1α, 28s rDNA, COI, COII, Cytb and 16s rDNA) were amplified through PCR in a total volume of 25 µL. The volume consisted of 12.5 µL CWBIO 2 × Taq MasterMix, 8.5 µL sterile distilled H2O, 2.0 µL genomic DNA template and 1.0 µL 10 µM each primer. The primers used and corresponding annealing temperature in PCR as well as references are listed in Table 2 . After electrophoretic analysis to ensure the amplification products were the target fragments we needed, the PCR products were subsequently sent to Sunny Biotechnology Co., Ltd. (Shanghai, China) for sequencing with the same primers used in the PCR. All sequences gathered in this study have been deposited in the GenBank.
3
Genomic DNA and RNA Extraction from Nematodes
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Genomic DNA was extracted from mixed-stage C. elegans (N2 strain) using the EasyPure Genomic DNA Kit (TransGen Biotech, China); total RNA was isolated using the TRIzol Plus Purification kit (Life Technologies, USA). Genomic DNA of H. contortus was extracted from cultured L3s, and total RNA was isolated from various stages, including eggs, first-stage larvae (L1), second-stage larvae (L2), infective L3s (iL3s), females and males of fourth-stage (L4) and adults using the same kits. RNA integrity and yields were verified by electrophoresis and spectrophotometric analysis (NanoDrop Technologies), respectively. RNA was treated with RQ1-RNase-Free DNase (Promega, USA). Nucleic acids were frozen and stored at -80°C.
4
Extracting DNA and RNA from Infective Larvae
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Genomic DNA was extracted from 10,000–20,000 iL3s (infective third-stage larvae) using an EasyPure Genomic DNA kit (TransGen Biotech, Beijing, China) and used immediately or stored at − 20 °C until use. Total RNA was extracted from ~ 25,000 iL3s by TRizol reagent extraction according to the manufacturer’s instructions (Life Technologies, USA). A complementary DNA (cDNA) was amplified using PrimeScript First-Strand cDNA Synthesis Kit (Takara, Beijing, China) and stored at − 80 °C until further analysis.
5
Quantification of Organellar Genome Copy Number
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Tachyzoites (iPYK1 or iPYK1-Δpyk2 strain) with or without 3 or 5 days’ ATc treatment were forced to egress by needle passage and purified for DNA extraction using the EasyPure genomic DNA kit (Transgen Biotech, Beijing, China). Thirty ng DNA from each sample was subject to qPCR analysis, using primers (listed in Table S1 ) designed to amplify fragments from the apicoplast genome (EF-Tu), mitochondrial genome (CytB), and nuclear genome (UPRT), respectively, as previously described (43 (link)– (link)45 (link)). The qPCR was performed using the Power SYBR green PCR master mix (Toyobo Co., Ltd., Osaka, Japan), and all reactions were performed on the ABI ViiA 7 detection system (Life Technologies, Inc., Rockville, MD, USA). The threshold cycle (CT) values for the nuclear genome amplification were used as a normalization reference to compare apicoplast and mitochondrial genome abundance across samples. The 2ΔΔCT method was used to estimate the changes of apicoplast and mitochondrial genome abundance after ATc treatment. Every sample was independently tested three times, each with two technical replicates.
6
Genomic DNA Methylation Analysis
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Genomic DNA from patient samples and cell lines was isolated using an Easypure Genomic DNA kit (TransGen Biotech, Beijing, China). We conducted a bisulfite conversion and purification using the EpiTect Bisulfite (QIAGEN, CA) with the procedure according to the product manual. Methylation-specific PCR (MSP) and bisulfite sequencing PCR (BSP) were performed and analyzed as previously described.21 (link),22 (link)
7
Isolation and Identification of Enterococcus faecium
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Enterococcus faecium isolates, E. coli strains and plasmids used in this study are listed in Table 1 . E. faecium were isolated from a previous study (Feng et al., 2016 (link)). E. faecium isolates were identified by 16s rRNA gene sequencing and subsequent blasting of the sequences, using the Basic Local Alignment Search Tool (BLAST) program. The genomic DNA was extracted from overnight culture using an EasyPure Genomic DNA kit (TransGen Biotech, Beijing, China) as per manufacturer’s protocol. A PCR reaction was performed to amplify the 16S rRNA genes using universal primers 27F and 1492R (Supplementary Table S1 ). PCR products were then sequenced using the Sanger sequencing. E. faecium isolates were routinely grown in the de Man, Rogosa, and Sharpe (MRS) broth or on the agar at 37°C. E. coli EC1000 (Leenhouts, 1996 (link)) was used for plasmid construction and was grown in the Luria-Bertani (LB) broth at 30°C. Where necessary, antibiotics were used at the following concentrations: chloramphenicol 5 μg/ml for E. faecium, spectinomycin 300 μg/ml for E. faecium, and 100 μg/ml for E. coli, ampicillin 100 μg/ml for E. coli. All antibiotics were obtained from Sigma (St. Louis, MO, United States).
8
Methylation Analysis of HUVEC Genomic DNA
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HUVECs genomic DNA was isolated with the Easypure Genomic DNA kit (TransGen Biotech, Beijing, China). After genomic DNA was isolated, the EZ DNA Methylation-Gold Kit (Zymo Research Corporation, CA, USA) was used for bisulfite conversion and purification according to protocol. Then, methylation-specific PCR (MSP) was performed for methylated and unmethylated forms by simultaneous use of primers. The positive controls in PCRs were performed with HUVECs, and the negative controls were performed with water, respectively. The bisulfite sequencing PCR (BSP) primer was designed by Sangon Biotech (Shanghai). pMD19-T (Takara, Beijing, China) was used for purifying and cloning of amplified PCR products. Five to ten clones of each cell were sequenced. CpG viewer, QUMA, and BIO-analyzer were used for comprehensively and comparatively calculating the percentage of methylation.
9
Isolation and Characterization of Insect 5-HT7 Receptor
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Genomic DNA and total RNA were extracted from adult M. separata using an EasyPure Genomic DNA Kit (Transgen Biotech, Beijing, China) and TRIzol reagent (Invitrogen, Carlsbad, CA, USA), respectively. Genomic DNA and single-stranded cDNA synthesized from the RNA using the Fast King gDNA Dispelling RT SuperMix (TianGen, Beijing, China) were used as the template for PCRs to amplify the genomic DNA sequences and open reading frame (ORF) of Msep5-HT7, respectively. Full-length sequences of putative Msep5-HT7 were obtained from our previous brain transcriptomes of M. separata [59 (link)]. The gene-specific primers (Table 4 ) used in our studies were designed by Primer Premier 5.0 software. The amplified DNA fragments were cloned into the pMD19-T Simple Vector (Takara, Dalian, China). Positive clones were identified and sequenced by Sangon Biotech (Shanghai, China). The obtained cDNA sequences were submitted to NCBI as Msep5-HT7L (GenBank Acc. OM025087) and Msep5-HT7S (GenBank Acc. OM025088).
10
Complete Mitochondrial Genome Sequencing
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Total genomic DNA was extracted from the muscle sample using the EasyPure® Genomic DNA Kit according to the manufacturer’s instructions (TransGen Biotech Co, Beijing, China). The integrity of the genomic DNA was assessed by 1% agarose gel electrophoresis and the concentration was assessed using NanoDrop (Thermo Fisher Scientific, Waltham, MA, USA). After DNA extraction and quality detection, a sample was used to sequence the complete mitochondrial genome, which was performed on the Illumina Novaseq 6000 platform (Illumina, San Diego, CA, USA) with PE 2 × 150 bp.
Partial sequences of the cox1 gene were amplified using the primer pairs F – 5′-ACTAATCAYAARGATATTGG-3′ and R – 5′-CCAGTAGGAAYAGCAATAAT-3′ modified from Chen et al. (2011 (link)). Each PCR reaction was performed using a total volume of 50 μl containing 1 μl (approximately 100 ng) of genomic DNA, 25 μl of 2 × EasyTaq PCR SuperMix (TransGen Biotech, Beijing, China), and 1 μl of 10 μmol/l forward and reverse primers. The PCR thermocycler program was as follows: 94 °C for 5 min as initial denaturalization, followed by 35 cycles of 94 °C for 30 s, 50 °C for 1 min, 72 °C for 2 min, and a final extension at 72 °C for 10 min. PCR products were purified and sequenced in both directions on an automatic sequencer (ABI PRISM 3730, Boray Biotechnology Co., Ltd., Xiamen, China).
Partial sequences of the cox1 gene were amplified using the primer pairs F – 5′-ACTAATCAYAARGATATTGG-3′ and R – 5′-CCAGTAGGAAYAGCAATAAT-3′ modified from Chen et al. (2011 (link)). Each PCR reaction was performed using a total volume of 50 μl containing 1 μl (approximately 100 ng) of genomic DNA, 25 μl of 2 × EasyTaq PCR SuperMix (TransGen Biotech, Beijing, China), and 1 μl of 10 μmol/l forward and reverse primers. The PCR thermocycler program was as follows: 94 °C for 5 min as initial denaturalization, followed by 35 cycles of 94 °C for 30 s, 50 °C for 1 min, 72 °C for 2 min, and a final extension at 72 °C for 10 min. PCR products were purified and sequenced in both directions on an automatic sequencer (ABI PRISM 3730, Boray Biotechnology Co., Ltd., Xiamen, China).
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