Model 3730xl automated dna sequencing system

The LAB strains isolated from kimchi were identified by 16S rRNA sequencing analysis. The cell stock was inoculated into MRS broth and incubated at 30 °C for 2 days. The cultures were sent to Macrogen (Seoul, Korea) for sequencing analysis. Genomic DNA of the LAB strains was amplified using two PCR primers (27F: 5′-AGAGTTTGATCMTGGCTCAG-3′; 1492R: 5′-TACGGYTACCTTGTTACGACTT-3′). The PCR product was purified using a Montage PCR Clean up kit (Millipore, Burlington, MA, USA). The purified PCR product was sequenced using two sequencing primers (85F: 5′-GGATTAGATACCCTGGTA-3′; 907R: 5′-CCGTCAATTCMTTTRAGTTT-3′). Sequencing was performed on the Big Dye Terminator Cycle Sequencing Kit v.3.1 (Applied BioSystems, Foster City, CA, USA). Sequencing products were resolved on an Applied Biosystems model 3730XL automated DNA sequencing system (Applied BioSystems, Foster City, CA, USA) at Macrogen. The 16S rRNA sequences of LAB strains were compared with data in GenBank using the Basic Local Alignment Search Tool (BLAST).

Detail information for the cloning and transformation has been previously published (Kim et al. 2012 (link)). The primers used to amplify the 16S rRNA genes for bacteria were 27F and 1492R (Lane 1991 ). The amplification conditions for the PCR were 95°C for 5 min, followed by 30 cycles of 95°C for 45 sec, 55°C for 45 sec, and 72°C for 90 sec, with a final extension step for 5 min at 72°C. PCR products were purified with a QIAquick PCR purification kit (Qiagen, Valencia, CA) and ligated into a pUC118 HincII/BAP vector (Takara Bio Inc.), which was transformed into competent Escherichia coli DH5α cells (Invitrogen Corp., Carlsbad, CA) using heat shock. Plasmids from E. coli DH5α transformants were isolated using the PureLink Quick Plasmid Miniprep kit (Invitrogen Corp.). The 16S rRNA genes from the bacterial clones were sequenced using an Applied BioSystems model 3730xl automated DNA sequencing system (Foster City, CA).

In August 2020, fungal macroforms were observed only in the La Concha orchard. Internal samples were obtained in the laboratory from two fruiting bodies of different colors, and DNA extraction was performed using a commercial kit (Ultraclean Isolation DNA Kit, MoBio brand, San Mateo, CA, USA) following the manufacturer’s instructions. The quality and concentration of the DNA were verified using a Nanodrop 2000c spectrophotometer. The internal transcribed spacer (ITS) gene region, 18S rDNA (partial sequence) gene, 5.8S rDNA gene, internal transcribed spacers 1 and 2 (full sequence), and the 28S rDNA gene (partial sequence) were amplified by PCR using the ITS5 (5′-GGAAGTAAAAGTCGTAACAAGG-3′) [42 ] and ITS4 5′-TCCTCCGCTTATTGATATGC-3′) primers [42 ].
The protocol included an initial denaturation at 94 °C for 1 min, followed by 40 cycles each of 94 °C (1 min), 50 °C (2 min), and 72 °C (1 min), and a final extension at 72 °C (5 min). PCR was conducted using a Peltier Thermal Cycler PTC-200 (Bio-Rad, Hercules, CA, USA), and the PCR products were verified by electrophoresis on a 1.2% agarose gel. PCR products were sequenced in both directions using an Applied Biosystems Model 3730XL Automated DNA Sequencing System.