At 24 h, 48 h, and 96 h after spraying, 50 S. litura larvae were collected from each plate and dissected to obtain the midgut region of each larva. The collected midguts were rinsed with sterile water and stored at -20 °C for DNA extraction. DNA was extracted using a bacterial DNA extraction kit (Omega Bio-Tek, USA) and dissolved in 200 μL of ddH 2 O. DNA concentration and purity were measured using 1% agarose gel electrophoresis and a nanodrop 1000 spectrophotometer (Saveen Werner ApS, Denmark), respectively. Extracted DNA was diluted to a concentration of 1 ng/μl and stored at -20 °C. The diluted DNA was used as a template for the amplification of bacterial 16S rRNA genes using polymerase chain reactions (PCRs) with barcoded primers and Takara Ex Taq (Takara Biomedical Technology (Beijing) Co., Ltd.). To analyze bacterial diversity, the V3-V4 variable regions of the 16S rRNA sequence were amplified using the universal primers 343F (5′-TACGGRAGGCAGCAG-3′) and 798R (5′-AGGGTATCTAATCCT-3′).
Bacterial dna extraction kit
Manufactured by Omega Bio-Tek
Sourced in United States
The Bacterial DNA Extraction Kit is a laboratory tool designed to efficiently extract and purify bacterial DNA from a variety of sample types. The kit utilizes a standardized protocol and reagents to facilitate the isolation of high-quality genomic DNA, which can then be used for downstream molecular biology applications.
Automatically generated - may contain errors
Lab products found in correlation
Takara ex taq, by Takara Bio (1 mentions)
Qubit dna assay kit, by Thermo Fisher Scientific (1 mentions)
Ddh2o, by Tiangen Biotech (1 mentions)
Fungal dna extraction kit, by Omega Bio-Tek (1 mentions)
Novaseq platform, by Illumina (1 mentions)
Primerquest tool, by Integrated DNA Technologies (1 mentions)
7 protocols using bacterial dna extraction kit
1
Analyzing Bacterial Diversity in Spodoptera litura
2
Microbial Genomic DNA Extraction
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Strains were cultured in mMRS liquid medium at 37 °C for 24–48 h and then pelleted for genomic DNA extraction. Genomic DNA was extracted using a bacterial DNA extraction kit (Omega Bio-Tek, Norcross, GA, USA) following the manufacturer’s instructions. The extracted genomic DNA was tested for quality using agarose gel electrophoresis (1% gel concentration), purity using a UV spectrophotometer, and concentration using a QubitTM 4 fluorometer and a Qubit DNA Assay Kit (Life Technologies, Carlsbad, CA, USA).
3
Bacterial 16S rRNA Gene Sequencing
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The total genomic DNA of bacterial isolates was extracted using the Bacterial DNA Extraction Kit (Omega Bio-Tek, Norcross, GA, USA) according to its manufacturer’s instructions. The 16S rRNA gene fragments of the isolates were amplified using the primers 27F and 1492R [28 ] with PCR conditions as described previously [26 (link)]. Amplicons were purified and sent to Qinke (Qinke company, Xi’an, China) for sequencing on an Applied Biosystems 3730XL sequencer (ABI 3730XL). The MEGA 11 tool was used to assemble the whole 16S rRNA gene sequences [29 (link)]. BLAST analysis of the 16S rRNA gene sequences using the EzTaxon database (EzBioCloud.net | Search about Bacteria or Archaea) was performed to obtain the top hits (described species) with type material sequencings [30 (link)]. The threshold value of 98.7% for 16S rRNA gene similarity was used to assess whether the isolates were potential new species [31 (link)].
4
Bacterial 16S rRNA Amplification and Identification
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The total genomic DNA of the bacterial isolates was extracted using the Bacterial DNA Extraction Kit (Omega Bio-Tek, Norcross, GA, USA) according to its manufacturer’s instructions. The 16S rRNA gene fragment was amplified using the primers 27F and 1492R [37 ]. The PCR reaction cycling condition was as follows: initial denaturation at 94 °C for 5 min, followed by 30 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, elongation at 72 °C for 45 s, and final elongation at 72 ℃ for 10 min. PCR products were purified and sent to Qinke (Qinke Company, Xi’an, China) for sequencing by the Applied Biosystems 3730XL (ABI 3730XL) sequencer. With the sequencing results, the strains were identified using the EzTaxon-e server [38 (link)]. The neighbor-joining trees were recovered in MEGA 6.0 [39 (link)]. The sequences were aligned with ClustalW [40 (link)]. Kimura’s two-parameters model was utilized to calculate evolutionary distances [41 (link)]. Confidence in the topologies of the resultant tree was calculated by the bootstrap test with 1000 resamplings [42 (link)].
5
Bacterial and Fungal DNA Extraction and Identification
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A DNA extraction of the isolates was conducted following the procedure specified by the manufactures of a bacterial DNA extraction kit (Omega Bio-tek, Inc., Morgan Hill, CA,USA) and a fungal DNA extraction kit (Omega Bio-tek, Inc., Morgan Hill, CA, USA). A 16S rDNA fragment was amplified by polymerase chain reaction (PCR) with 27F (5'-AGA GTT TGA TCC TGG CTC AG-3') and 1492R (5'-GGT TAC CTT GTT ACG ACT T-3'). An internal transcribed spacer (ITS) rDNA fragment was amplified by ITS1 (5'-TCC GTA GGT GAA CCT GCG G-3') and ITS4 (5'-TCC TCG CCT TAT TGA TAT GC-3'). The PCR was a 50 μL system, including template DNA 2 μL, forward primer 2 μL,reverse primer 2 μL, 2 × mastermix 25 μL, and DdH2O 19 μL (Tiangen, Beijing, China). The conditions for PCR were as follows: 95 °C for 5 min in initial denaturation, 35 cycles of 95 °C for 30 s, 55 °C for 35 s, 72 °C for a 2 min denaturation annealing and extension, and 72 °C for a 10 min final extension of the amplified DNA. The PCR products were checked for the expected size on 1% agarose gel and were sequenced at Huada Gene Company (Beijing, China). The sequences were compared against the GenBank database using the NCBI BLAST program. Phylogenetic trees were constructed using MEGA 5.0 software (National Institutes of Health, Bethesda, MD, USA). The sequences were deposited into GenBank and the accession numbers were obtained.
6
Whole-Genome Sequencing and Annotation of E. coli
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Whole-genome sequencing was performed on all strains. Briefly, single colonies were grown for 16 h in LB broth at 37 °C and bacterial DNA was extracted using a bacterial DNA extraction kit (Omega Bio-Tek, USA) according to the manufacturer’s instructions. Whole-genome sequencing was performed by Novegene (Novogene BioTech, Beijing, China) on an Illumina Novaseq platform. Genomes were assembled using Shovill version 1.0.4 [23 (link)] and annotated using Prokka software [24 (link)]. Single nucleotide polymorphism (SNP) analysis was performed using Snippy [25 (link)]. The online tool DAVID [26 (link)] was used to annotate SNP genes according to biological function and cellular components via Gene Ontology analysis [27 (link)]. The genome of the E. coli strains were submitted to GenBank under accession number PRJNA907472.
7
Genomic Screening of Biocontrol Bacillus
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The genome of cold-tolerant biocontrol B. atrophaeus strain TS1 was screened for the presence of genes responsible for the production of extracellular enzymes and siderophores. The genomic DNA was extracted from bacterial cells harvested from 24 h of grown culture in LB medium. A Bacterial DNA Extraction Kit (Omega Bio-tek, Norcross, GA, USA) was used to extract DNA by using the manufacturer’s guidelines. The gene sequences from Bacillus model strain 168 were retrieved from NCBI and searched in the TS1 genome by using local alignment tools. The primers were synthesized using the PrimerQuest tool of Integrated DNA Technologies (IDT). A DNA Master Mix (Vazyme Biotech. Co. Ltd., Nanjing, China) was used to amplify the targeted genes by using the reaction mixture and PCR profile provided by the manufacturer. The PCR primers used in this are listed in Table S2 .
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