Cells were spun down at 4200g for 10 minutes and the supernatant was removed. Pellets were resuspended in 100μL of PBS and lysozyme (Sigma #L6876) was added to reach a final concentration of 1000U/mL. Plates were sealed and vortexed until the suspension was homogenous (at least 2 minutes), then incubated at 37°C overnight. To each well, 2μL of 20ug/μL proteinase K (NEB, #P8107S) and 2μL of 0.8% SDS solution were added, and plates were then incubated at 55°C for an additional 3 hours, after which the cell suspensions had no apparent turbidity. DNA was purified from these cell lysates using the standard protocol in the DNA extraction kits (Zymo Research #D3010), except that DNA was resuspended in sterile water in the final step. DNA was quantified using SYBR Safe (Thermo, #S33102) and normalized the concentration of each sample to between 1 and 5 ng/μL. To prepare libraries for Illumina sequencing, the tagmentation-based plexWell DNA library prep kits (Seqwell, #PW096) was used according to the manufacturer’s protocol.
Dna extraction kit
Manufactured by Zymo Research
Sourced in United States
The DNA extraction kit is a lab equipment product designed to isolate and purify DNA from a variety of sample types. The kit contains the necessary reagents and components to effectively extract DNA, making it a useful tool for various scientific applications that require high-quality DNA samples.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop 2000, by Thermo Fisher Scientific (6 mentions)
Platinum taq hi fidelity polymerase, by Thermo Fisher Scientific (2 mentions)
Miseq 100, by Illumina (2 mentions)
De man rogosa sharpe broth, by Thermo Fisher Scientific (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Nanodrop one, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Sybr safe, by Thermo Fisher Scientific (1 mentions)
Proteinase k, by New England Biolabs (1 mentions)
Lysozyme, by Merck Group (1 mentions)
Luria bertani broth, by Merck Group (1 mentions)
Anaerogen system, by Thermo Fisher Scientific (1 mentions)
Quick dna fungal bacterial miniprep kit, by Zymo Research (1 mentions)
Sybr green pcr mix, by Thermo Fisher Scientific (1 mentions)
Lb broth, by Merck Group (1 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (1 mentions)
Pcr thermal cycler, by Thermo Fisher Scientific (1 mentions)
Proteinase k, by Merck Group (1 mentions)
T100 cycler, by Bio-Rad (1 mentions)
Deionized water, by Merck Group (1 mentions)
26 protocols using dna extraction kit
1
DNA Extraction and Illumina Library Prep
2
DNA Extraction from P. aeruginosa
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All multidrug resistant P. aeruginosa isolates were subcultured overnight in Luria-Bertani broth (Merck, Germany) and DNA was extracted from typical colonies of P. aeruginosa strains using Zymo research DNA extraction kits (Irvine, CA, USA), according to manufacturer’s instructions.
3
Infant Gut Microbiome Profiling
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Infant stools were collected from diapers and aliquoted within 24 hours into sterile tubes and stored at −80° C. DNA extraction was then conducted using Zymo DNA extraction kits (Tustin, CA, USA) on thawed samples, followed by quantity and purity assessment using OD260/280 nanodrop measurement (19 (link)). Fusion primers were used to obtain 16S ribosomal RNA (rRNA) V4-V5 amplicons. One of eight five-nucleotide long forward primers was used to connect the Illumina-specific bridge and sequencing primer regions with the 16S region, whereas the reverse primer was simply one of 12 Illumina indices.
16S rRNA amplification of the V4-V5 hypervariable regions was conducted at the Marine Biological Laboratory in Woods Hole, Massachusetts, USA for microbial profiling using both forward and reverse primers (20 (link), 21 (link)). Due to the use of both forward and reverse primers, 96 samples were multiplexed per lane. PCR was then conducted using triplicate samples of 33 μL, and a combination of 1.0 U Platinum Taq Hi-Fidelity Polymerase (Life Technologies Carlsbad, CA, USA), 1X Hi-Fidelity buffer, 200 μM dNTP3 PurePeak DNA polymerase mix (Pierce Nucleic Acid Technologies Milwaukee, WI, USA), 1.5 mM MgSO4 and 0.2 μM of each primer. Lastly, qPCR was used to quantify each library pool before sequencing using paired-end Illumina MiSeq 100 cycle runs.
16S rRNA amplification of the V4-V5 hypervariable regions was conducted at the Marine Biological Laboratory in Woods Hole, Massachusetts, USA for microbial profiling using both forward and reverse primers (20 (link), 21 (link)). Due to the use of both forward and reverse primers, 96 samples were multiplexed per lane. PCR was then conducted using triplicate samples of 33 μL, and a combination of 1.0 U Platinum Taq Hi-Fidelity Polymerase (Life Technologies Carlsbad, CA, USA), 1X Hi-Fidelity buffer, 200 μM dNTP3 PurePeak DNA polymerase mix (Pierce Nucleic Acid Technologies Milwaukee, WI, USA), 1.5 mM MgSO4 and 0.2 μM of each primer. Lastly, qPCR was used to quantify each library pool before sequencing using paired-end Illumina MiSeq 100 cycle runs.
4
Infant Gut Microbiome Profiling
5
Inverse PCR for Gene Disruption in Drosophila
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Inverse PCR was performed by following the GDP_iPCR Protocol from the Gene Disruption Project (Bellen et al. 2004 (link)). Briefly, genomic DNA was extracted from the whole body (DNA extraction kit; Cat #D6015; Zymo Research) and cut with Hinp I. DNA fragments were ligated as PCR templates. A pair of primers EY.3.F and EY.3.R was used (http://flypush.imgen.bcm.tmc.edu/pscreen/files/GDP_iPCRProtocol_051611.pdf ). The sequence of PCR product was blasted online against the Drosophila melanogaster genome sequence.
6
Feline Blood DNA Archive Profiling
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The DNA archive of the Royal Veterinary College (RVC) was searched for feline ethylenediaminetetraacetic acid (EDTA) blood samples that were residuals from previous routine hematology testing. Cats were selected to represent the widest age range possible based on the available samples with a uniform distribution across the entire range, available breeds, and neutering status. As the samples originated from cats that were presented for veterinary investigation, cats were selected to have no or minimal abnormalities on available laboratory data (hematology, serum biochemistry, endocrinology), reviewed by a board certified veterinary clinical pathologists (BSz). The DNA samples were maintained frozen at − 80 °C for various amount of time (0–11 years). Samples from guinea pigs, rabbits, ferrets, and alpacas were also residual samples from routine patients presented for veterinary care. Sample collection was approved by the Clinical Research Ethical Review Board of the RVC (URN: 2019 1947–2). Genomic DNA from cat blood was extracted using the Zymo DNA extraction kit according to the manufacturer’s instructions. DNA was eluted in water and quantified with picogreen kit according to the instructions provided.
7
Gut Microbiome DNA Extraction Protocol
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Total genomic DNA was extracted from all fecal samples using bead-beating and a DNA extraction kit (Zymo Research Corp, USA) according to the manufacturer's protocol. The DNA concentration and purity were assessed using a NanoDrop spectrophotometer (Thermo Scientific, USA). The DNA samples were adjusted to 5 ng/μl and used for high-throughput 16S sequencing and quantitative polymerase chain reaction (qPCR) analyses.
8
Listeria DNA Extraction and PCR Amplification
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The ZYMO DNA extraction kit was used to extract DNA from presumptive Listeria spp. colonies. For amplification, a partial fragment of the prs gene was targeted on PCR using the primer sequence prs-F (5′-GCT GAA GAG ATT GCG AAA GAA G-3′) prs-R (5′-CAA AGA AAC CTT GGA TTT GCG G-3′) we used that amplified a fragment of 370 bp [28 (link)]. Briefly, One Taq® Quick-Load® from Biolabs and 50 µL reactions of PCR were run. We used a PCR master mix consisting of 25 µL One Taq Quick-load 2X Master Mix with Standard Buffer, 1 uL of 10 µM of each forward and reverse primer, 20 µL of nuclease-free water, and 3 µL of the template. We used thermocycling conditions where initial denaturation was at 94 °C for 1 min, followed by 30 cycles at 94 °C for 45 s, annealing at 53 °C for 45 s, and extension at 72 °C for 2 min, with a final extension step at 72 °C for 5 min. Finally, the expected PCR product band size of 370 bp was visualized on 1.5% agarose gel coated with ethidium bromide.
9
Isolation and Characterization of Probiotic Bacterial Strain
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The candidate probiotic bacterial strain was isolated from the gastrointestinal tracts of compassionately sacrificed weaned piglets of the indigenous South African Windsnyer pig breed (APIEC13/008). The organism was cultured in de Man-Rogosa-Sharpe broth (Oxoid, UK), under strict anaerobic conditions and incubated at 37°C for 24 hours in an anaerobic jar provided with an AnaeroGen system (Thermofisher, UK). The culture was later washed twice in phosphate buffer saline (PBS) and centrifuge at 6000 rpm for 5 minutes each time. Bacterial genomic DNA was extracted with a DNA extraction kit (Zymo Research, USA), in accordance with the manufacturer’s instructions. The degree of purity and concentration of the extracted DNA were determined using a nanodrop spectrophotometer (NanoDrop 2000, ThermoFisher). The genomic DNA material was kept at −20°C for further use.
10
Extraction and Quantification of Bacterial DNA
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DNA was extracted from the presumptive STEC colonies on CHROMagar using Zymo Research DNA extraction kit, Quick-DNA™ Fungal/Bacterial Miniprep Kit (D6005) according to manufacturer’s instruction. The DNA was then quantified using a NanoDrop spectrophotometer (NanoDrop one, Thermo Scientific).
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