The genomic DNA of selected LAB strains, potential producers of therapeutic biomolecules, was extracted using a Maxwell® DNA Cell kit in a Maxwell® 16 Research System instrument (Promega, Madison, WI, USA). WGSwas conducted on an Illumina MiSeq 2500 platform (Illumina, San Diego, CA, USA) at IGA Technology Services (IGA Technology Services Srl, Udine, Italy) using a paired-end approach [53 (link)]. This Whole Genome Shotgun project was submitted to the NCBI nt database (BioProject PRJNA388578, version 1.0) under the accession numbers listed in the Table 4 for each strain with the following feature: biosynthesis of S-layer proteins, plantaricin and EPSs [22 ].
Maxwell dna cell kit
Manufactured by Promega
Sourced in United States
The Maxwell® DNA Cell kit is a laboratory equipment product designed for automated DNA extraction from a variety of cell types. It provides a streamlined and efficient method for isolating high-quality DNA samples.
Automatically generated - may contain errors
3 protocols using maxwell dna cell kit
1
Genomic DNA extraction and WGS of LAB strains
2
Genomic Sequencing and Annotation of L. fermentum D12
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Genomic DNA of L. fermentum D12 strain was extracted using a Maxwell® DNA Cell kit in a Maxwell® 16 Research System instrument (Promega, USA). Sequencing was performed on the Illumina MiSeq 2500 (Illumina, San Diego, CA) at IGA Technology Services (Udine, Italy) using a paired-end approach, as described in Banić et al. [25 (link)]. Contigs were classified as belonging to L. fermentum D12 when obtaining the best BLASTn v2.2.27 hit [46 (link)] in the NCBI nt database, where the whole genome sequence was submitted. The genome of L. fermentum D12 was uploaded to the web annotation service Rapid Annotations using Subsystems Technology (RAST; http://rast.nmpdr.org/rast.cgi ) for automated annotation of sequenced genes, including those involved in EPS biosynthesis, followed by manual scanning [47 (link)]. This Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the Accession no. RHMA01000000 (BioProject PRJNA388578, Biosample SAMN10319712). The version described in this paper is the first one. A circular map of the L. fermentum D12 genome was created using the DNA Plotter [48 (link)]. Furthermore, a BLASTp search against the NCBI protein (nr) database was performed.
3
Gut Lactobacillus Analysis in Rats
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PCR-DGGE analysis performed according to Leboš Pavunc et al. (2012) with slight modi cations in order to check the presence of the Lb. plantarum SF9B and Lb. brevis SF9C in faeces of Lactobacillus fed rats. DNA was extracted directly from faecal samples of healthy rats for culture-independent PCR-DGGE analysis, as well as from the bacterial colonies, isolated on MRS agar plates for culture-dependent PCR-DGGE analysis, from faeces of healthy rats sampled before feeding (control), and 3 rd and 10 th day after application of Lactobacillus SF9B and SF9C strains. In both cases, DNA was isolated using Maxwell DNA Cell Kit with automated extraction platform, Maxwell® 16 Research System instrument (Promega, USA). The V2-V3 region of the 16S ribosomal DNA gene of bacteria in the faeces contents or from pure cultures of lactobacilli was ampli ed with primers HDA1-GC and HDA2.
To identify the lactobacilli, recovered from rat faeces, the V2-V3 region of the 16S rRNA gene of the strains was ampli ed. The amplicons were sequenced using ABI PRISM ® 3100-Avant Analyzer (Applied Biosystems). A search of sequences deposited in the GenBank DNA database was conducted by using the BLAST algorithm. The of the isolates were determined based on the highest score.
To identify the lactobacilli, recovered from rat faeces, the V2-V3 region of the 16S rRNA gene of the strains was ampli ed. The amplicons were sequenced using ABI PRISM ® 3100-Avant Analyzer (Applied Biosystems). A search of sequences deposited in the GenBank DNA database was conducted by using the BLAST algorithm. The of the isolates were determined based on the highest score.
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