Genomic DNA of the mutants which showed reduced virulence on cabbage were purified by using a Nucleospin Microbial DNA Kit (TaKaRa, Ohtsu, Japan) and digested with Hind III, Xh I, Sph I, Kpn I, Sal I, Xba I, or Hinc II (TaKaRa). The resultant DNA was ligated with T4 DNA ligase (Ligation-convenience kit, Nippon Gene, Tokyo, Japan), then introduced into E. coli DH5α competent cells. Plasmid DNA was purified from the transformants, and transposon-insertion sites were identified by sequencing with the M13 forward primer. A Pseudomonas Genome DB BLAST search (http://www.pseudomonas.com/blast/setnblast ) was utilized to identify the function of the mutated genes (Fig. 1 ).
Nucleospin microbial dna kit
Manufactured by Takara Bio
Sourced in Japan, United States
The Nucleospin Microbial DNA Kit is a laboratory equipment designed for the efficient extraction and purification of microbial DNA from a variety of sample sources. It utilizes a silica membrane-based method to capture and elute high-quality genomic DNA.
Automatically generated - may contain errors
Lab products found in correlation
Hindiii, by Takara Bio (2 mentions)
Xbai, by Takara Bio (2 mentions)
Hinc 2, by Takara Bio (2 mentions)
Miseq platform, by Illumina (2 mentions)
T4 dna ligase ligation convenience kit, by Nippon Gene (1 mentions)
T4 dna ligase, by Nippon Gene (1 mentions)
Xhoi, by Takara Bio (1 mentions)
Qubit 4 fluorometer, by Thermo Fisher Scientific (1 mentions)
Nextera xt index kit v2, by Illumina (1 mentions)
Herculase 2 fusion dna polymerase, by Agilent Technologies (1 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Takara la taq dna polymerase, by Takara Bio (1 mentions)
7 protocols using nucleospin microbial dna kit
1
Identifying Virulence Genes in Pseudomonas Mutants
2
Mutant Virulence Reduction in Kiwifruit
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Genomic DNA of the mutants that showed reduced virulence on kiwifruit was purified using a Nucleospin Microbial DNA Kit (Takara Bio, Kusatsu, Shiga, Japan) and digested with Hind III, Xho I, Sph I, Kpn I, Sal I, Xba I, or Hinc II (Takara Bio). The resultant DNA was ligated with T4 DNA ligase (Ligation-convenience kit, Nippon Gene, Tokyo, Japan), then introduced into E. coli DH5α competent cells. Plasmid DNA was purified from the transformants, and transposon-insertion sites were identified by sequencing with the M13 forward primer. A Pseudomonas Genome DB BLAST search (http://www.pseudomonas.com/blast/setnblast , accessed on 14 February 2020) was utilized to identify the function of the mutated genes.
3
Genomic DNA Extraction from Enterococcus faecium
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Enterococcus faecium LR13 preserved as glycerol stock (50% v/v) at −80°C in our laboratory was revived by incubating overnight in deMan, Rogosa and Sharpe (MRS) broth at 37°C, 200 rpm. Bacteria were grown up to the exponential phase (OD600 = 0.8) and harvested by centrifugation at 8,000 rpm for 8 min at 4°C. Genomic DNA was extracted using Nucleospin Microbial DNA kit (Takara Bio, San Jose, CA, USA) following the manufacturer’s instructions. DNA concentrations were determined using Qubit 4 fluorometer (Thermo Fisher Scientific, Waltham, MA, USA).
4
Metagenomic analysis of gut microbiome in mice
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Fresh fecal samples were collected from five wild-type or ASC−/− mice, and genomic DNA was extracted using the NucleoSpin Microbial DNA Kit (TaKaRa, cat. no. U0235) according to the manufacturer’s instructions. The extracted genomic DNA was sent to Macrogen (Tokyo, Japan) for metagenomics sequencing. The sequencing library was prepared by random fragmentation of the DNA, followed by 5′ and 3′ adapter ligation using Herculase II fusion DNA polymerase (Agilent, cat. no. 600675) and Nextera XT Index Kit V2 (Illumina, cat. no. FC-131-1001), according Illumina’s guidelines for 16S metagenomic sequencing library preparation (Illumina, Part #15044223 Rev. B). The amplicon of 16S rRNA was sequenced using paired-end sequencing on the Illumina MiSeq platform. The sequences were clustered de novo into operational taxonomic units (OTUs) using a CD-HIT-EST-based OTU analysis program (CD-HIT-OTU) and a cutoff of >97% similarity. The Shannon diversity index and the Inverse Simpson index, which account for both richness and evenness, and in which an increased value indicates greater diversity, were used to compare the microbial diversity of the OTUs in the libraries. The PCoA analysis of the weighted UniFrac distances was applied to the distance matrices for visualization of the microbial diversity of the OTUs in the libraries.
5
Microbial Diversity in Senmaizuke Pickles
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Bacterial cells were collected from the pickled juice of senmaizuke samples via centrifugation. The cells were disrupted using zirconia beads, and genomic DNA was extracted and purified using the NucleoSpin Microbial DNA kit (TakaraBio). Sequencing analysis using an Illumina MiSeq platform was conducted by Bioengineering Lab. Co., Ltd. (Kanagawa, Japan). The V3/V4 region of the 16S rRNA gene was targeted. The sequences derived from plant chloroplast were excluded in the data analysis. The Shannon index (microbial diversity) was calculated using a previously described equation (Spellerberg & Fedor, 2003 (link)).
6
Bacterial Strain Isolation and Genomic DNA Extraction
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Twenty-nine culture were selected for the experiment. Among them, 24 type strains were provided from the Korean Agricultural Culture Collection (KACC) and five strains of P. odoriferum isolated from the Republic of Korea obtained from KACC and the Highland Agriculture Research Institute to validate the specificity of the species-specific primers developed in this study (Table 1 ).
The bacterial isolates were cultured at 28–30°C for 24–48 h on trypsin soy agar media (15 g/l digested casein, 5 g/l digested soy, 5 g/l NaCl, and 1.5% agar, Difco, Detroit, MI, USA) under aerophilic condition. The stock culture were preserved in glycerol stocks at −80°C. Total genomic DNA was extracted using the NucleoSpin Microbial DNA Kit (TaKaRa Bio USA, Inc., San Jose, CA, USA). The corresponding strains listed inTable 1 and isolated DNA was spectrophotometrically quantified. The concentration and quality of the extracted genomic DNA were determined using a NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). All the extracted genomic DNA samples were stored at −20°C until used for further experiments.
The bacterial isolates were cultured at 28–30°C for 24–48 h on trypsin soy agar media (15 g/l digested casein, 5 g/l digested soy, 5 g/l NaCl, and 1.5% agar, Difco, Detroit, MI, USA) under aerophilic condition. The stock culture were preserved in glycerol stocks at −80°C. Total genomic DNA was extracted using the NucleoSpin Microbial DNA Kit (TaKaRa Bio USA, Inc., San Jose, CA, USA). The corresponding strains listed in
7
Actinomycete 16S rRNA Sequencing Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Genomic DNA was extracted from actinomycete cells and purified using the NucleoSpin Microbial DNA kit (Takara Bio Inc., Shiga, Japan). PCR amplification of the 16S rRNA gene was performed using universal bacterial primers and TaKaRa LA Taq DNA polymerase (Takara Bio). The sequences of the primers used in this study were as follows: Bac27 (forward), 5ʼ-AGAGTTTGATC (A/C) TGGCTCAG-3ʼ; Bac1492 (reverse), 5ʼ-GGTTACCTTGTTACGACTT-3ʼ 20) . DNA sequencing was performed at the Central Laboratory of Osaka Metropolitan University Medical School.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!