A propidium monoazide (PMA, Biotium, Inc., Hayword, CA) treatment was applied to the homogenized and filtered yacon samples as described by Pan and Breidt (2007) (link) prior to DNA extraction. Total genomic DNA was extracted using the PowerSoil DNA isolation kit (MO BIO Laboratories Inc., Carlsbad, CA) following the manufacturer’s instructions. DNA from isolated colonies growing on MRS was extracted using the InstaGene Matrix DNA extraction kit (Bio-Rad, Hercules, CA) following the manufacturer’s instructions.
Instagene matrix dna extraction kit
Manufactured by Bio-Rad
Sourced in United States
The InstaGene Matrix DNA extraction kit is a laboratory product designed to efficiently extract DNA from various sample types. It utilizes a simple and rapid method to purify DNA without the need for organic solvents or centrifugation steps.
Automatically generated - may contain errors
Lab products found in correlation
S1000, by Bio-Rad (3 mentions)
Phorbol myristate acetate (pma), by Biotium (1 mentions)
Powersoil dna isolation kit, by Qiagen (1 mentions)
Egg yolk and tellurite, by Beijing Land Bridge Technology (1 mentions)
Baird parker agar, by Beijing Land Bridge Technology (1 mentions)
Taq dna polymerase 2 master mix red, by Ampliqon (1 mentions)
O generuler 1 kb dna ladder, by Thermo Fisher Scientific (1 mentions)
Emeraldamp max pcr master mix kit, by Takara Bio (1 mentions)
5 protocols using instagene matrix dna extraction kit
1
DNA Extraction from Yacon Samples
2
Isolation and Identification of Staphylococcus aureus
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To isolate and detect the S. aureus, each sample was diluted 10-fold in sterile peptone water and homogenized. Aliquots (1 mL) were placed onto Baird-Parker agar supplemented with 5% egg yolk and tellurite (Beijing Land Bridge Technology Ltd., Beijing, China). The plates were incubated for 24–48 h at 37°C. Colonies with typical black appearance and surrounded by a clear zone were enumerated as S. aureus.
Presumptive colonies were confirmed by polymerase chain reaction (PCR) (Bio-Rad S1000, United States) detection of the thermonuclease gene (nuc, S. aureus specific) (Supplementary Table 1 ). DNA of the strains was extracted using the InstaGene Matrix DNA extraction kit (Bio-Rad Laboratories, Hercules, California, United States) following the manufacturer’s instructions. The amplification conditions and reagents for the PCR assays were those described by Liu et al. (2017) (link). Negative control (without DNA template) and positive control (S. aureus ATCC 6538) templates were included in all PCR assays. After identification, one to three colonies per sample were randomly selected for subsequent analysis. All strains were stored with sterile magnetic beads at −80°C until further analysis.
Presumptive colonies were confirmed by polymerase chain reaction (PCR) (Bio-Rad S1000, United States) detection of the thermonuclease gene (nuc, S. aureus specific) (
3
Yeast DNA Extraction and Rep-PCR Profiling
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The total yeast DNA was extracted from the colonies grown on MYGP agar plates using the InstaGene Matrix DNA extraction kit (Bio-Rad Laboratories, Hercules, CA, USA). Rep-PCR was conducted in a 25 μL volume mixture containing 13 μL of Taq DNA Polymerase 2× Master Mix RED (Ampliqon, Odense, Denmark), 5 μL Primer GTG5 (Integrated DNA Technologies, Denmark), 4 μL sterile Milli-Q water, and 3 μL DNA. The PCR reaction was carried out in a SureCycler 8800 thermocycler (Agilent Technologies, Santa Clara, CA, USA) using the following program: initial denaturation for 7 min at 95 °C, followed by 30 cycles of 95 °C for 1 min, 45 °C for 1 min, and 65 °C for 8 min, and an elongation step of 65 °C for 16 min. The rep-PCR products were separated by 1.5% agarose gel electrophoresis (5 h, 120 V) in Tris-Borate-EDTA buffer (0.5 × TBE), using an O’GeneRuler 1 kb DNA ladder (Thermo Scientific, Roskilde, Denmark) as a reference marker. The rep-PCR profiles were clustered using Bionumerics 7.1 software (Applied Maths, BioMérieux, Schaerbeek, Belgium) based on Dice’s Coefficient of similarity with the Unweighted Pair Group Method and Arithmetic mean clustering algorithm (UPGMA).
4
Isolation and Identification of B. cereus
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For B. cereus sl isolation, all the samples were diluted and first chosen according to the National Food Safety Standard Method for Food Microbiological Examination (GB 4789.40-2010) (Gao et al., 2018 (link)). In total, 166 colonies have been picked. These 166 colonies were picked and then placed onto the Mannitol-Yolk Polymyxin Agar Plate (Beijing Land Bridge Technology Co. Ltd., Beijing, China), and then cultured at 30°C for 24 h (Owusu-Kwarteng et al., 2017 (link)). The colonies with pink color (81/166, 48.80%) were selected for further identification.
Identification of B. cereus sl isolates used a polymerase chain reaction (PCR) thermal cycler (Bio-Rad S1000; Bio-Rad, Hercules, CA, USA) via 16S rRNA and panC gene. DNA of B. cereus isolates was extracted with the InstaGene Matrix DNA Extraction Kit (Bio-Rad) following the instructions of Owusu-Kwarteng et al. (2017 (link)). The panC gene analysis was performed using the primers (Sangon, China), and conditions are shown inSupplementary Table S1 with negative and positive (DNA of B. cereus ATCC 11778; China Center of Industrial Culture Collection, Beijing, China) controls.
The PCR products were sequenced by SinoGenoMax Co. Ltd. Then, the sequencing results were BLASTed in the GenBank database (http://www.ncbi.nlm.nih.gov/BLAST/ ). The degree of homology for the universal 16S rRNA gene and the panC gene was higher than 99%.
Identification of B. cereus sl isolates used a polymerase chain reaction (PCR) thermal cycler (Bio-Rad S1000; Bio-Rad, Hercules, CA, USA) via 16S rRNA and panC gene. DNA of B. cereus isolates was extracted with the InstaGene Matrix DNA Extraction Kit (Bio-Rad) following the instructions of Owusu-Kwarteng et al. (2017 (link)). The panC gene analysis was performed using the primers (Sangon, China), and conditions are shown in
The PCR products were sequenced by SinoGenoMax Co. Ltd. Then, the sequencing results were BLASTed in the GenBank database (
5
Staph. aureus Identification by PCR
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Presumptive colonies were confirmed by PCR detection (Bio-Rad S1000, Bio-Rad Laboratories, Hercules, CA) of the Staph. aureus-specific thermonuclease gene (nuc; Supplemental Table S1, https:// doi .org/ 10 .3168/ jds .2017 -13370). All isolates were stored at -80°C until use.
Genomic DNA of the Staph. aureus isolates was extracted using the InstaGene Matrix DNA extraction kit (Bio-Rad Laboratories) following the manufacturer's instructions. The PCR reactions were performed using the EmeraldAmp Max PCR Master Mix kit (Takara, Dalian, China) and followed the manufacturer's instructions. Briefly, 25-µL reactions were prepared, containing 12.5 µL of 2× EmeraldAmp Max PCR Master Mix kit (Takara), 1 µL of extracted DNA, 10 pmol of each primer, and ultrapure water (Takara). The amplification conditions were as follows: 94°C for 1 min; 37 cycles of 94°C for 1 min, 55°C for 30 s, and 72°C for 90 s; and 72°C for 3.5 min for a final extension step. Negative controls (without DNA template) and a positive control (Staph. aureus ATCC 6538 template) were included in all PCR assays.
Genomic DNA of the Staph. aureus isolates was extracted using the InstaGene Matrix DNA extraction kit (Bio-Rad Laboratories) following the manufacturer's instructions. The PCR reactions were performed using the EmeraldAmp Max PCR Master Mix kit (Takara, Dalian, China) and followed the manufacturer's instructions. Briefly, 25-µL reactions were prepared, containing 12.5 µL of 2× EmeraldAmp Max PCR Master Mix kit (Takara), 1 µL of extracted DNA, 10 pmol of each primer, and ultrapure water (Takara). The amplification conditions were as follows: 94°C for 1 min; 37 cycles of 94°C for 1 min, 55°C for 30 s, and 72°C for 90 s; and 72°C for 3.5 min for a final extension step. Negative controls (without DNA template) and a positive control (Staph. aureus ATCC 6538 template) were included in all PCR assays.
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