Fresh bacterial cultures on nutrient agar plates were used for the DNA extraction. The DNA of bacteria was extracted with the Zymo DNA extraction kit (Zymo Research, Irvine, CA, USA; Cat. No. D6005) as specified in the manufacturer’s instructions. A NanoDrop spectrophotometer was, thereafter, used to determine extracted DNA samples’ concentrations at a 260 nm wavelength; agarose gel electrophoresis was used to determine the presence of DNA. Also, 10 μL of Hyper Ladder™ 1 kb and 4 μL of bacterial DNA samples were run in 1.0% agarose concocted through the heating of 1 g of agarose powder in 100 mL of 1 × TAE at 65 °C that lasted 4 min, followed by the addition of 10 μL of ethidium bromide. The bands of DNA were observed in a gel Doc machine.
Zymo dna extraction kit
Manufactured by Zymo Research
Sourced in United States
The Zymo DNA extraction kit is a laboratory product designed to efficiently extract and purify DNA samples from a variety of biological sources. It utilizes a streamlined process to isolate high-quality DNA for further analysis and experimentation.
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Lab products found in correlation
10 protocols using zymo dna extraction kit
1
Bacterial DNA Extraction and Quantification
2
Infant Stool Microbiome Extraction
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Infant stool samples were collected at the 6-week maternal postpartum follow-up appointment and were aliquoted and frozen at − 80 °C within 24 h. Following established methods reviewed by Wu et al. [30 (link)], we used the Zymo DNA extraction kit (Zymo Research) to extract microbial DNA from thawed samples and quantified the DNA using OD260/280 nanodrop.
3
Infant Gut Microbiome Analysis Protocol
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We measured the fecal microbiome of infant stool as a measure of the infant gut microbiome. Infant stool samples were collected at regularly scheduled ~6-week postpartum follow-up appointments as described previously13 (link),27 (link). Samples were aliquoted and frozen at −80 °C within 24 h of receipt. A Zymo DNA extraction kit (Zymo Research) was used for DNA extraction from thawed samples, and an OD260/280 nanodrop was used to measure sample quality and purity. The V4-V5 hypervariable region of the bacterial 16S rRNA gene was sequenced using Illumina MiSeq at the Marine Biological Laboratory in Woods Hole, MA. Amplicon sequence variants (ASVs) were then inferred using DADA228 (link), and taxonomies were assigned using the SILVA database29 (link). Quality control measures were conducted as described previously13 (link). A subset of stool samples was also sequenced with NGS and shotgun metagenomics sequencing as previously described21 (link). Extracted DNA samples were sheared to a mean insert size of 400 bp using a Covaris S220 focused ultrasonicator. The sequencing libraries were constructed using Nugen’s Ovation Ultralow V2 protocol, and samples were sequenced using Illumina NextSeq. DNA reads were merged and trimmed using KneadData30 (link) for quality control before species-level taxonomic profiles were generated using Metaphlan231 (link).
4
Infant Gut Microbiome Profiling
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We collected infant stool samples at approximately 6 weeks and 12 months of age.10 (link),34 (link) These samples were aliquoted and frozen at −80°C, and DNA was extracted from thawed samples using Zymo DNA extraction kit (Zymo Research, Irvine, CA). OD260/280 nanodrop was used to measure sample quality and purity. Samples were sent to Marine Biological Laboratory in Woods Hole, Massachusetts, for bacterial 16S rRNA gene sequencing of the V4-V5 hypervariable region using Illumina MiSeq (Illumina, San Diego, CA). We amplified samples in triplicate with one negative control for quality control.10 (link) We inferred amplicon sequence variants (ASVs) using DADA230 (link) and assigned taxonomies using the SILVA database.29 (link)
5
Infant Gut Microbiome Analysis Using 16S rRNA Sequencing and Metagenomics
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16S Microbiome Profiling of Infant Stool
7
Breastmilk Collection and Processing Protocol
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Breast milk samples were collected at home by study participants from unsterilized bilateral breasts, with separate study-provided sterile collection bottles used for milk from each breast. To capture a representative portrait of infant exposure during breastfeeding we did not use a sterile collection protocol. Subjects provided a minimum of 18 mL and up to 80 mL of milk from each breast, with a median of 35 mL per breast. Samples were stored in the refrigerator at participants homes for up to approximately 1 day, brought in cold packs to the postpartum follow-up appointment (between 3.7 and 12 weeks after delivery), and immediately chilled. Samples were processed within 24 h of receipt. Before sample processing, the milk was mixed by inverting the collection tubes 10 times. 3 mL of milk from each breast were combined, centrifuged at 3396 rcf for 90 min at 4°C. Fat and supernatant were removed, and 150 μL of PBS was added to and mixed with the cell pellet. The cell suspension was transferred to a 1.5 mL tube and frozen at −20°C. The sample was thawed at room temperature, and microbial DNA was extracted using the Zymo DNA extraction kit (Zymo Research) and was quantified using the OD260/280 Nanodrop.
8
Bacterial DNA Extraction and Quality Assurance
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Extraction of the genomic DNA from bacterial cultures was done using a Zymo DNA extraction kit (Zymo Research, USA) following the manufacturer's protocol. This was carried out at the Biotechnology Center of the Biotechnology and Nuclear Agricultural Research Institute of the Ghana Atomic Energy Commission. The eluted DNA was stored at −20OC until further analysis. The quality of the extracted DNA was determined using 1% agarose gel and viewed under a high performance ultraviolet transilluminator (UVP, Cambridge, UK).
9
Molecular Identification of Seed-Borne Fungi
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The molecular technique based on the Polymerase Chain Reaction (PCR) was used to confirm identity of selected seed-borne fungal isolates. From 7-day-old cultures, 100 mg of mycelium was scraped and DNA was isolated using Zymo DNA extraction kits (Zymo Research, USA) following the manufacturer's protocol. Primer pair ITS 1F and ITS 4R were used to amplify the Internal Transcribed Spacer (ITS1 and 2) conserved regions (White et al. 1990) . Each 50μl reaction mixture included 21 μL of PCR-grade water, 1 μL of DNA template, 1.5 μM of each primer, and 1 μL of PCR Master Mix (2X) (0.25 μL Taq DNA polymerase, reaction buffer, 4 mM MgCl2 and 0.4 mM of each dNTP; Thermo Scientific, Waltham, USA). The PCR conditions consisted of a denaturation step at 94 °C for 2 min, followed by 35 cycles at 94 °C for 1 min, 55 °C for 30 s, 72 °C for 1 min and a final elongation step at 72 °C for 10 min. The amplified DNA was purified using a Zymo purification kit (Inqaba Biotech, South Africa), concentration was measured using a NanoDrop 1000 Spectrophotometer (Thermo Scientific, Wilmington, DE, USA) and adjusted to 50 ng/μL. 2).
10
Molecular Identification of Seed-Borne Fungi
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The molecular technique based on the Polymerase Chain Reaction (PCR) was used to confirm identity of selected seed-borne fungal isolates. From 7-day-old cultures, 100 mg of mycelium was scraped and DNA was isolated using Zymo DNA extraction kits (Zymo Research, USA) following the manufacturer's protocol. Primer pair ITS 1F and ITS 4R were used to amplify the Internal Transcribed Spacer (ITS1 and 2) conserved regions (White et al. 1990) . Each 50μl reaction mixture included 21 μL of PCR-grade water, 1 μL of DNA template, 1.5 μM of each primer, and 1 μL of PCR Master Mix (2X) (0.25 μL Taq DNA polymerase, reaction buffer, 4 mM MgCl2 and 0.4 mM of each dNTP; Thermo Scientific, Waltham, USA). The PCR conditions consisted of a denaturation step at 94 °C for 2 min, followed by 35 cycles at 94 °C for 1 min, 55 °C for 30 s, 72 °C for 1 min and a final elongation step at 72 °C for 10 min. The amplified DNA was purified using a Zymo purification kit (Inqaba Biotech, South Africa), concentration was measured using a NanoDrop 1000 Spectrophotometer (Thermo Scientific, Wilmington, DE, USA) and adjusted to 50 ng/μL. 2).
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