DNA was extracted from 0.4 g soil taken from each layer of the soil cores obtained at −99 h, −3 h, 3 h, 24 h, and 69 h of incubation (n = 3) using a FastDNA spin kit for soil (MP Biomedical, Santa Ana, CA, USA). To remove humic substances capable of potentially inhibiting enzymatic reactions in downstream analyses65 (link), additional purification was performed using a MicroSpin S-400 HR column (GE Healthcare, Little Chalfont, UK) and a DNA clean & concentrator-5 kit (Zymo Research, Irvine, CA, USA). The concentrations of extracted DNA were measured using a NanoDrop ND-1000 UV-Vis spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).
Microspin s 400 hr column
Manufactured by GE Healthcare
Sourced in United Kingdom, United States
The MicroSpin S-400 HR columns are size-exclusion chromatography columns designed for the purification and separation of biomolecules such as proteins, peptides, and nucleic acids. The columns feature a high-resolution matrix that allows for efficient separation of molecules based on their size and molecular weight.
Automatically generated - may contain errors
Lab products found in correlation
Minelute pcr purification kit, by Qiagen (2 mentions)
Dna clean concentrator 5 kit, by Zymo Research (1 mentions)
Fastdna spin kit for soil, by MP Biomedicals (1 mentions)
Nanodrop nd 1000 uv vis spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Biotin teg, by Merck Group (1 mentions)
Ultracentrifuge, by Beckman Coulter (1 mentions)
Novaseq platform, by Illumina (1 mentions)
T3 polymerase, by Promega (1 mentions)
γ 32p atp, by MP Biomedicals (1 mentions)
T4 polynucleotide kinase, by New England Biolabs (1 mentions)
Fastdna spin kit for soil, by Qbiogene (1 mentions)
Dna clean and concentrator 25 kit, by Zymo Research (1 mentions)
Takara ex taq, by Takara Bio (1 mentions)
Lambda dna, by Merck Group (1 mentions)
3730xl dna analyzer, by Thermo Fisher Scientific (1 mentions)
Gotaq hot start polymerase, by Promega (1 mentions)
Nucleospin gel and pcr clean up, by Macherey-Nagel (1 mentions)
Pgem t easy vector, by Promega (1 mentions)
Dnazol direct, by Molecular Research Center (1 mentions)
2500 platform, by Illumina (1 mentions)
23 protocols using microspin s 400 hr column
1
Soil DNA Extraction and Purification
2
In vitro Reconstitution of RIDA Complex
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The RIDA complex was assembled similarly as described previously (31 (link)) with several modifications. The first reaction mixture containing β-clamp (25 pmol), the clamp loader (132 fmol) and ϕX174 open circular form (0.5 pmol) were incubated in buffer RIDA-β [20 mM Tris/HCl (pH 7.5), 10% (v/v) glycerol, 8 mM DTT, 0.01% (w/v) Brij-58, 8 mM Magnesium acetate, 20 mM potassium l -glutamate, 2 mM ATP] for 20 min at 32°C (final volume: 25 μl). The nucleoprotein complex was isolated by molecular filtration (0.63 ml MicroSpin S-400 HR column, GE Healthcare) in void volume. The second reaction mixture containing Hda (0.31 pmol) and 2 mM ADP was preincubated for 10 min at 32°C. The third reaction mixture contained DnaA (0.5 pmol), ATP (2 mM), cardiolipin (14 μg/ml), which was incubated for 10 min at 37°C; followed by the incubation with fluphenazine (0.15 mM) for 10 min at 37°C. The reaction mixtures were mixed together, incubated for 10 min and the nucleoprotein complex was isolated by molecular filtration as previously. The RIDA complex assembly was resolved in 12.5% SDS-PAGE, followed by silver staining.
3
Chromatin Assembly with Yeast Histones
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Yeast histones (6.86 μg), Nap1 (14.61 μg) and ISW1A (384 ng) were mixed in a 40 μL reaction volume in buffer containing 10 mM HEPES-KOH pH 7.5, 50 mM KCl, 5 mM MgCl2, 0.5 mM EDTA, 10% glycerol and 0.1 mg/ml BSA and incubated for 30 minutes on ice. Then, 45 mM creatine phosphate, 3 mM ATP, 6 μg creatine phosphate kinase and 1 μg plasmid DNA were added and incubated at 30°C for 5 hours. Following chromatin assembly, the buffer was exchanged to 10 mM HEPES-KOH pH 7.5, 50 mM KCl and 10% glycerol using an illustra MicroSpin S-400 HR Column (GE Healthcare).
4
Biotin-labeled DNA Nanostructure Assembly
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To bind the SA, 3′ end of a staple strand (5′-CGA AGC ACT CAT TTT TGG GAA CTG GAG TTA TCC CTA TTT TTT CCT GAA GTA C) at the corner of square 5 of SnL was modified with BiotinTEG and was purchased from Sigma-Aldrich. The monomeric Sn for dimers and 1D chains were assembled with staple strand with biotin and purified according to the method described above. The assembled dimers and 1D chains from the 10 nM monomeric Sn were mixed with SA at a ratio of 1 : 3 and incubated 1 h at room temperature. Then, the excess SA was removed using the Microspin S-400 HR column (GE Healthcare Life Sciences) following the manufacturer's protocol.
5
Purification and Fractionation of Plant Ribosomes
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The total ribosomes and polysomes were prepared as previously described (51 ), with the following modifications. Actively growing leaf samples (25 g) were washed with DEPC-treated water, frozen in liquid nitrogen and macerated to a fine powder using a cold mortar and pestle. The resulting powder was then homogenized in 2 volumes of cold plant extraction buffer (50 mM Tris–HCl pH 9.0, 30 mM MgCl2, 400 mM KCl, 17% [w/w] sucrose) and clarified by filtering through DEPC-treated Miracloth cheesecloth. The resulting extracts were centrifuged at 1000 × g for 7 min at 4°C. One tenth of a volume of 20% Triton X-100 was added to the supernatants, and they were then centrifuged at 15 500 × g for 20 min. The clear supernatants were then carefully layered on 60% (w/v) sucrose cushions (20 mM Tris–HCl pH 7.6, 5 mM MgCl2, 510 mM NH4Cl, 60% (w/v) sucrose) and centrifuged at 140 000 × g for 19 h using a SW28 rotor in a Beckman Coulter ultracentrifuge. The resulting pellets were carefully rinsed with resuspension buffer (50 mM KCl, 20 mM Tris–HCl pH 7.6, 5 mM MgCl2), and were then resuspended in 200 μl of the same buffer.
For further ribosome purification by gel filtration (52 (link)), MicroSpin S-400 HR columns were used according to manufacturer's instructions (GE Healthcare UK).
For further ribosome purification by gel filtration (52 (link)), MicroSpin S-400 HR columns were used according to manufacturer's instructions (GE Healthcare UK).
6
NS5A Resistance Mutation Detection
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The nested PCR products were purified using the Microspin™ S-400 HR Columns, GE Healthcare, Buckinghamshire, UK) according to the manufacturer’s instructions. DNA sequencing was performed using the Big Dye® Terminator v 3.1 Cycle Sequencing Kit with a DXL 3500 DX sequencer (Appied Biosystem, Courtaboeuf, France). The same primers used in the two semi-nested PCR for the amplification of the NS5A region for both subtypes 1a and 1b were used for sequencing in this region.
Nucleotide sequences were aligned with reference sequences of different subtypes with SeqScape v 2.7 (Applied Biosystem, Courtaboeuf, France) and analyzed for the presence of previously identified mutations conferring resistance to NS5A inhibitors. GeneBank accession numbers for NS5A reference sequences are AF009606 for subtype 1a and AY045702 for genotype 1b. The threshold of nucleotide mixture detection during sequencing of samples is estimated to be around 20 %.
Nucleotide sequences were aligned with reference sequences of different subtypes with SeqScape v 2.7 (Applied Biosystem, Courtaboeuf, France) and analyzed for the presence of previously identified mutations conferring resistance to NS5A inhibitors. GeneBank accession numbers for NS5A reference sequences are AF009606 for subtype 1a and AY045702 for genotype 1b. The threshold of nucleotide mixture detection during sequencing of samples is estimated to be around 20 %.
7
Ribosome Profiling of BEAS-2B Cells
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A total of 1×107 BEAS-2B cells were lysed in polysome lysis buffer (20 mM Tris•Cl at pH 7.4, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 1% Triton X-100, Turbo DNase I 25 U/ml, 50 mg/mL cycloheximide). Then, cell lysate was treated with unspecific endoribonuclease RNase to digest the free RNAs other than ribosome-protected fragments (RPFs). Isolation of ribosome was performed by size-exclusion chromatography with MicroSpin S-400 HR columns (GE Healthcare, USA). Recovered RPFs were then subjected to rRNA depletion kit to remove rRNA contamination. Small RNA (< 200 bp) was resolved by electrophoresis on a 15% (w/v) polyacrylamide gel (PAGE) with 7 M urea. The RPFs bands (~ 30 nt) were cut for RNA extraction, phosphorylated and ligated with 5′ and 3′ adapters, reverse transcribed and PCR amplified, respectively. Libraries were then sequenced on Illumina Novaseq platform using the PE150 (paired-end 150 nt) recipe. The sequencing reads were mapped to human genome database by TopHat2 software and then quantified by the HTSeq and DESeq2 R packages.
8
Ribosome Profiling with Monosome Purification
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Ribosome profiling was performed as earlier described (22 (link)), with the following modifications. Monosomes were purified using Microspin S-400 HR columns (GE Healthcare 27-5140-01) and 15–35 nt ribosome-protected fragments were isolated for library generation.
9
Virucidal Activity of Surfactants and Ternary Mixtures
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200 μL of the viral stock solution was added to 200 μL of surfactant (C12E8 and/or [DiC10][Cl]) or 200 μL of the aqueous ternary mixtures of CD, C12E8 and [DiC10][Cl] (all solutions were in equimolar proportions). After incubation for 15 min (or 2 h) at 25 ± 0.1°C, the mixtures were immediately filtered on MicroSpin S-400 HR columns (GE Healthcare) to separate viruses from the other components of the mixtures. Residual viruses were then titrated as described above. Each experiment was performed at least twice. The virucidal activity was determined by the difference of the logarithmic titer of the virus control minus the logarithmic titer of the test virus. This difference is presented as a reduction factor including its 95% confidence interval. A reduction in virus titer of ≥4-log10 was regarded as evidence of sufficient virucidal activity according to EN 14476. The lowest concentration giving a reduction in virus titer of ≥4-log10 was the minimum virucidal concentration (MVC).
10
Lycorine Disinfection Assay for CHIKV
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To exclude the possibility of degermation of lycorine, a disinfectant test was designed. Briefly, equal volumes of eGFP-CHIKV (2 × 104 PFU in 100 μL) and 10 μmol/L of lycorine were mixed and incubated at 37 °C for 1 h. The MicroSpin S-400 HR columns (GE Healthcare) were used to trap the lycorine before inoculation into Vero cells. At 24 hpi, the eGFP expression was captured under a fluorescence microscope.
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