Bacterial cells were grown at 37 °C in 500 mL of TSB broth until reaching the late exponential phase (OD590 = 1), and then harvested via centrifugation at 5000× g for 30 min at 4 °C. High-molecular-weight genomic DNA was purified using a cetyl trimethyl ammonium bromide (CTAB)-based method [24 (link)]. Briefly, a cell pellet was dry vortex-mixed and lysed for 15 min at 37 °C in a lysis solution containing sodium dodecyl sulphate (SDS) 0.008% and sodium deoxycholate (DOC) 0.1%, as described in [25 (link)]. Proteins and polysaccharides were precipitated in 0.5 M NaCl and CTAB/NaCl (10% CTAB, 0.7 M NaCl) at 65 °C for 10 min. High-molecular-weight DNA was purified three times with 1 volume of chloroform/isoamyl alcohol (24:1 (v:v)), precipitated in 0.6 volumes of ice-cold isopropanol and spooled on a glass rod. DNA was resuspended in 10-fold diluted saline-sodium citrate (SSC) 1× buffer, adjusted to 1× SSC, homogenized using a rotator mixer and stored at 4 °C. DNA was quantified with a Qubit 3.0 Fluorometer (Invitrogen, Waltham, MA, USA) using the Qubit dsDNA BR Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) and with a spectrophotometer measurement (Implen, Munich, Germany). DNA integrity and size were assayed via horizontal gel electrophoresis using 0.6% Seakem LE (Lonza, Rockland, ME, USA) agarose in 0.5× Tris Borate EDTA running buffer.
Seakem le
Manufactured by Lonza
Sourced in United States, Switzerland
SeaKem LE is a laboratory equipment product manufactured by Lonza. It is a high-performance agarose gel designed for a wide range of electrophoresis applications. The product's core function is to provide a reliable and consistent matrix for the separation and analysis of nucleic acids.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Qubit 3.0 fluorometer, by Thermo Fisher Scientific (1 mentions)
Qubit dsdna br assay kit, by Thermo Fisher Scientific (1 mentions)
Oragene collection kit, by DNA Genotek (1 mentions)
Sybr safe dna gel stain, by Thermo Fisher Scientific (1 mentions)
Chloroquine, by Merck Group (1 mentions)
Positively charged nylon membrane, by Roche (1 mentions)
Sybr gold nucleic acid gel stain, by Thermo Fisher Scientific (1 mentions)
Primestar gxl dna polymerase, by Takara Bio (1 mentions)
Sybr safe, by Thermo Fisher Scientific (1 mentions)
Pulsed field certified agarose, by Bio-Rad (1 mentions)
Chef dr 3, by Bio-Rad (1 mentions)
Kapa taq dna polymerase, by Roche (1 mentions)
Kapa hifi hotstart readymix, by Roche (1 mentions)
13 protocols using seakem le
1
High-Molecular-Weight Bacterial DNA Extraction
2
DNA Extraction Methods and Integrity Verification
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The UCT IRD registry and biobank has archived biological material from patients for three decades; therefore, various DNA extraction methods have been used over the years. Blood samples were collected in EDTA tubes and processed immediately, mainly using a salting out method [36 (link)], although different commercial DNA extraction kits have been trialled for short periods at various times. Alternatively, the buffy coat was removed from the blood sample and frozen, and it was later processed using the salting out method. Since 2013, saliva samples have been collected using Oragene® collection kits (DNA Genotek Inc, Ottawa, Canada) and processed according to the manufacturers’ instructions.
DNA samples were quantified using the Nanodrop ND-1000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and subsequently diluted to a final concentration of 20 ng/µL in Sabax water (Adcock Ingram, Johannesburg, SA). After dilution, the DNA integrity was confirmed using 1–2% weight/volume (w/v) agarose gels containing 0.5–1 g agarose (Lonza SeaKem® LE, Basel, Switzerland), 50 mL of 1X Tris Borate EDTA (TBE) buffer and 5 µL SYBR® Safe DNA Gel Stain (Thermo Fisher Scientific).
DNA samples were quantified using the Nanodrop ND-1000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and subsequently diluted to a final concentration of 20 ng/µL in Sabax water (Adcock Ingram, Johannesburg, SA). After dilution, the DNA integrity was confirmed using 1–2% weight/volume (w/v) agarose gels containing 0.5–1 g agarose (Lonza SeaKem® LE, Basel, Switzerland), 50 mL of 1X Tris Borate EDTA (TBE) buffer and 5 µL SYBR® Safe DNA Gel Stain (Thermo Fisher Scientific).
3
2D Agarose Gel Electrophoresis for DNA Structure Analysis
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The first dimension was in a 0.4% agarose (Seakem®LE, Lonza) gel in TBE buffer (89 mM Tris borate, 2 mM EDTA) at 0.9 V/cm at room temperature for 25 h. The second dimension was in a 1% agarose gel in TBE buffer and was run perpendicular to the first dimension. The dissolved agarose was poured around the excised agarose lane from the first dimension and electrophoresis was at 5 V/cm in a 4°C cold chamber for 12 h. When necessary, different concentrations of chloroquine (Sigma) were added to the TBE buffer in both the agarose gel and the running buffer in the first dimension. Southern transfer was performed as described before [26 (link)–28 (link)].
4
Expelling Packaged DNA for Gel Electrophoresis
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To expel packaged DNA before gel electrophoresis, we added the following to a 12 µL portion of an undialyzed fraction of a Nycodenz density gradient: (1) 5 µL of 0.1 M NaCl, 0.01 M Tris-Cl, pH 7.4, 0.001 M ethylenediaminetetraacetic acid (EDTA) and (2) 3 µL of 30% sucrose, 0.6 M NaCl, 0.06 M Tris-Cl, pH 7.4, 0.06 M EDTA, 6% Sarkosyl NL97, 1.2 mg/mL bromophenol blue. This mixture was then incubated at 85 °C for 10 min. For gel electrophoresis, a portion of this mixture was layered in a sample well of a submerged 0.7% agarose (Seakem LE, Lonza Group, Rockland, ME, USA) gel. This gel had been cast in and submerged under 0.05 M sodium phosphate, pH 7.4, 0.001 M EDTA. We then performed electrophoresis in this buffer at 0.5 V/cm, 25 °C for 26.0 h. After electrophoresis, we stained nucleic acid in the gel by soaking the gel for 2 h. at room temperature (25 ± 2 °C) in 1:10,000 diluted GelStar (New England Biolabs, Ipswich, MA, USA) in 0.002 M sodium EDTA, pH 7.4. We destained the gel in 0.002 M sodium EDTA, pH 7.4 for at least 2 h. We photographed the gel during top-illumination with a Fotodyne #3-3000 transilluminator.
5
Two-Dimensional Agarose Gel Electrophoresis
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The first dimension was in a 0.4% Seakem®LE agarose (Lonza Rockland, Inc., Rockland, ME, USA), gel in TBE buffer (89 mM Tris-Borate, 2 mM EDTA) at 0.9 V/cm at room temperature for 25 h. The second dimension took place in a 1% agarose gel in TBE buffer run perpendicular to the first dimension. The dissolved agarose was poured around the excised agarose lane from the first dimension, and electrophoresis occurred at 5 V/cm in a 4 °C cold chamber for 10 h. Southern transfer was performed by washing the gels for 15 min in 0.25 N HCl before an overnight transfer to positively charged nylon membranes (Roche, Basel, Switzerland) in 0.4 N NaOH.
6
PCR amplification of Rickettsia traA-traH genes
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Genomic DNA from ISE6 cells, either uninfected or infected with A. phagocytophilum ApSheep_NorV1mutants, was obtained as described above. Fifty ng of genomic DNA was used as template in 50 µL reactions that contained 200 μM dNTPs, 0.25 μM of primers targeting rickettsial traA, traB, traD, traG, and traH genes (Table 3 ), and 1.25 units of PrimeSTAR GXL DNA polymerase (Takara). PCR amplification was performed in a BioRad PTC-100 thermal cycler (BioRad) with the following conditions: 94 °C 1 min, followed by 30 cycles at 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 1 min, and a final extension cycle at 72 °C for 5 min. PCR products were separated electrophoretically using a 1% Seakem LE (Lonza, Morristown, NJ, USA) agarose gel, and stained with SYBR Gold nucleic acid gel stain (ThermoFisher scientific, Waltham, MA, USA) for visualization.
7
Yeast and Bacterial Chromosome Preparation
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Plug preparation for yeast standards was performed according to described methods by Hage and Houseley49 (link). Bacterial plug preparation was performed similar, with the following alterations: Cultures were grown overnight at 30 °C with 200 rpm. An equivalent of 1 mL OD600 = 5 was harvested and used for plug preparation. The concentration of low melting agarose (SeaKem LE, Lonza) for plug preparation was reduced to 0.8%, and lysozyme (1 mg/mL) was used instead of lyticase. PFGE was undertaken by running samples on a 0.8% agarose gel using Pulsed-Field Certified Agarose (Bio-Rad) in 1× TAE buffer at 14 °C on a Bio-Rad clamped homogeneous electric field apparatus (CHEF-DR III, Bio-Rad). 3 V/cm were used with 46 h switch time of 600 s at 120 °C. The resulting gel was stained with 1× SYBR Safe (ThermoFisher Scientific) and imaged using Typhoon RGB laser scanning system. The known karyotypes of S. cerevisiae and S. pombe served as size standards.
8
CLEC4M Gene VNTR Polymorphism Genotyping
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The VNTR polymorphism in exon 4 of the CLEC4M gene was genotyped using the following PCR protocol: one cycle at 94°C for 3 min, followed by 10 cycles of 94°C for 15 s, 68°C for 30 s and 68°C for 1 min, then followed by 30 cycles at 94°C for 15 s, 67°C for 30 s and 68°C for 1 min, before a final extension cycle at 68°C for 10 min. The PCR reactions contained: 0.125 mM KAPA Taq DNA polymerase (KAPA Biosystems, Cape Town, South Africa), 1X KAPA Taq Extra Buffer, 1.75 mM MgCl2, 0.3 mM deoxyribonucleotide triphosphates, 2 ng/μl template DNA and 0.4 mM of each primer (S1 Table ). The PCR products were visualized with gel electrophoresis on 30 x 40 cm 3% agarose gels (Seakem LE, Lonza, Rockland, ME, USA) using separation for 2 h at 10 V/cm. The genotyping results were validated in several ways: 1) The proper inheritance of alleles was confirmed by the analysis of three sets of trios (mother, father and child), 2) control populations C1 and C2 were used to confirm that the overall allele frequencies were as expected for background populations compared with previous studies, and 3) the type 1 VWD population was analyzed twice and evaluated independently by two persons. A few uncertainties were resolved by repeated analysis.
9
Two-Dimensional Agarose Gel Electrophoresis
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The first dimension was in a 0.4% agarose gel (Seakem® LE; Lonza) in TBE buffer (89 mM Tris-borate, 2 mM ethylenediaminetetraacetic acid) at 0.9 V/cm at room temperature for 24–27 h. The second dimension was in a 1–1.2% agarose gel in TBE buffer and was run perpendicular to the first dimension. The dissolved agarose was poured around the excised agarose lane from the first dimension and electrophoresis was at 4.5 V/cm in a 4°C cold chamber for 10–12 h. in the presence of 0.3 μg/ml ethidium bromide. Southern transfer was performed as described before (38 (link),39 (link)).
10
Norovirus Detection via RT-PCR and Sequencing
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The samples that were positive for NoVs by rRT-PCR were subjected to reverse transcription PCR (RT-PCR) reactions, performed with the QIAGEN One-Step RT-PCR kit reagents (QIAGEN). Four different primer pairs that targeted the polymerase (ORF1) and/or the capsid (ORF2) region were used (Online Resource 1). Amplified products were visualized on 1.5 % SeaKem LE (Lonza, Basel, Switzerland) agarose gel with ethidium bromide staining, and sequenced according to the Sanger sequencing method in the Institute of Biotechnology, University of Helsinki, Finland.
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