Nucleosome sliding assays were conducted similar to Patel et al. (2011) (link). Remodeling reactions (20 µl) were performed at room temperature with 50 nM of either Chd1 or Chd1(W793A) and 150 nM FAM-labeled 80-601-0 nucleosomes in 1x Slide buffer (20 mM Hepes-KOH, pH 7.6, 50 mM KCl, 5 mM MgCl2, 0.1 mg/ml BSA, 1 mM DTT, 5% sucrose). After removal of a t = 0 time point, time-keeping was started upon addition of 2.5 mM ATP, with 1 µl of remodeling reaction transferred to 20 µl quench buffer (20 mM Hepes-KOH, pH 7.6, 50 mM KCl, 0.1 mg/ml BSA, 1 mM DTT, 5% sucrose, 5 mM EDTA, pH 8.0, and 150 ng/µl ultrapure salmon sperm DNA (Thermo Fisher Scientific, cat#15632011)) and tubes placed on ice. Time point samples were loaded onto 6% (60:1) acrylamide:bis-acrylamide native PAGE gels and run at 100V in 0.25xTBE for 100 min. Gels were scanned on a 9410 Typhoon variable mode imager (GE) and quantitated using ImageJ software (RRID:SCR_003070 ). Using Mathematica (RRID:SCR_014448 ), data were fit to the equation y = a1*(1-exp(-k1*x)) + a2*(1-exp(-k2*x))+c, where k1 and k2 are apparent rate constants, a1 and a2 are corresponding amplitudes, and c is a constant.
Ultrapure salmon sperm dna
Manufactured by Thermo Fisher Scientific
Sourced in United States
UltraPure Salmon Sperm DNA is a high-quality, highly purified DNA product derived from salmon sperm. It is suitable for use in various molecular biology applications, such as PCR, cloning, and transfection experiments.
Automatically generated - may contain errors
Lab products found in correlation
Ultrapure bsa, by Thermo Fisher Scientific (2 mentions)
E coli trna, by Merck Group (2 mentions)
Typhoon 9410 variable mode imager, by GE Healthcare (1 mentions)
α 32p dctp, by PerkinElmer (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Amersham hybond xl membrane, by GE Healthcare (1 mentions)
Quikhyb hybridization solution, by Agilent Technologies (1 mentions)
Bcabest labeling kit, by Takara Bio (1 mentions)
Xhoi, by New England Biolabs (1 mentions)
Hybond, by Cytiva (1 mentions)
Maxisorp elisa plate, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Jackson ImmunoResearch (1 mentions)
Tmb peroxidase substrate, by LGC (1 mentions)
Biophotometer plus, by Eppendorf (1 mentions)
Qiaamp dna mini blood mini kit, by Qiagen (1 mentions)
Sybr gold nucleic acid gel stain, by Thermo Fisher Scientific (1 mentions)
Hygromycin b, by GoldBio (1 mentions)
Infinite 200 pro microplate reader, by Tecan (1 mentions)
Yeast trna, by Thermo Fisher Scientific (1 mentions)
16 protocols using ultrapure salmon sperm dna
1
Nucleosome Sliding Assay for Chromatin Remodeling
2
Southern Blot Analysis of Genomic DNA
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Nuclear gDNA was extracted from liver tissue as indicated above. Then, gDNA was digested overnight with XhoI (NEB) that does not cut in the vector, but in the host gDNA. Digested DNA was run in a 1% TAE agarose gel at room temperature overnight. After electrophoresis, the gel was washed with denaturing buffer (3 M NaCl and 400 mM NaOH) twice for 5 min, and DNA was transferred to an Amersham Hybond-XL membrane (GE Healthcare) using transfer buffer (3 M NaCl and 8 mM NaOH) overnight. Membranes were washed with 2× saline sodium citrate (SSC) buffer for 5 min and blocked with UltraPure Salmon Sperm DNA (Thermo Fisher) in QuikHyb Hybridization Solution (Agilent Technologies) for 1 h at 65 °C. Probes for FLuc (500 bp) were generated using gel-purified PCR amplicons containing GFP sequence and a BcaBEST Labeling kit (Takara) and [α-32P]-dCTP (PerkinElmer), and probe hybridization were performed overnight at 65 °C with rotation. The membrane was washed with 2× SSC buffer and with 2× SSC containing 0.1% SDS at 65 °C. Signals were visualized using a Personal Molecular Imager System (Bio-Rad ChemiDoc Imaging Systems (vS.2.1 build 11)) and analyzed with Quantity One 1-D software v4.6.8 (Bio-Rad).
3
FISH Technique for Genetic Analysis
4
FISH Technique for Genetic Analysis
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When no immunofluorescence was performed prior to FISH, cells were fixed in 4% paraformaldehyde and permeabilized in 0.2% Triton X-100 as described above. When FISH was performed after immunofluorescence, no additional permeabilization step was performed. Cells were dehydrated sequentially in 70%, 95% and 100% ethanol for 5 minutes each. After a two minute drying step, cells were rehydrated in 2x SSC (1x SSC is 150 mM NaCl, 15 mM sodium citrate dihydrate, pH 7.0)/50% formamide (molecular biology grade) for 5 min. Slides were pre-hydrated in pre-hybridization solution (50% formamide, 2x SSC, 0.5 mg/mL UltraPure Salmon sperm DNA (ThermoFisher Scientific), 1 mg/mL UltraPure BSA (ThermoFisher Scientific), 0.13 mg/mL E. coli tRNA (Sigma Aldrich), 1 mM Vanadyl ribonucleoside complexes solution (Signal Aldrich) and 100 mg/mL dextran sulfate in ultrapure water) for 30 min at 37°C. After pre-hybridization, samples were hybridized with the probe (Supplementary Table 13 ) in pre-hybridization solution overnight at 37°C. On the following day, samples were washed twice in 2x SSC/50% formamide for 30 minutes each. Slides were rinsed in PBS once and sealed.
5
Microbiome-Specific Antibody Detection Assay
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Human gut commensal bacteria, comprising 33 commensal bacteria strains (RePOOPulate)56 (link), double-stranded DNA (UltraPure Salmon Sperm DNA, Thermo Fisher) and bovine serum albumin (BSA, Sigma-Aldrich) were coated on a MaxiSorp ELISA plate (Nunc) in PBS in triplicates and incubated overnight at 4 °C as recently reported56 (link). Plates were washed and blocked with 3% BSA in PBS for 2 h at RT before incubation with plasma (1:100) or CSF (1:5) for 1 h. After incubation with anti-human IgG or IgA horseradish peroxidase (Jackson ImmunoResearch) for 1 h, the assay was developed with TMB peroxidase substrate (Seracare). A polyclonal polyreactive IgG antibody (ED-38; undiluted transfection supernatant) was used as a positive assay control67 (link). Triplicates with a coefficient of variation (CV) greater than 15% were corrected for by excluding one value. Corrected duplicates with a CV above 15% were excluded from the analysis (n = 4). Negative control signals (secondary antibodies only) were subtracted in a plate-specific manner.
6
Isolation and Quantification of cfDNA from Mouse Plasma
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For isolation of cfDNA from mouse plasma, the Qiagen QIAamp DNA Mini Blood Mini Kit was used according to the manufacturer’s instructions (Qiagen, Valencia, CA, USA) after spinning plasma at 16,800×g for 10 min. A spectrophotometer (Eppendorf BioPhotometer Plus, Hamburg, Germany) was used to quantify levels of cfDNA. The optical density (OD) reflects the amount of DNA present and was measured at excitation and emission wavelengths of 260 and 280 nm, respectively. The OD/260 nm values were converted to nanogram per microliter using the conversion of 1 OD/260 nm = 50 ng/μL DNA. A standard calibration curve was obtained using known ssDNA concentrations (UltraPure Salmon Sperm DNA, ThermoFisher Scientific, Burlington, ON, Canada) ranging from 0 to 10 mg/mL and was used to confirm the accuracy of cfDNA concentrations calculated using the OD method.
7
Quantifying Plasma cfDNA via SYBR Gold Stain
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Plasma cfDNA was analyzed by the fluorescent SYBR™ Gold nucleic acid gel stain (Cat# S11494, Thermo Fisher Scientific, Waltham, MA, USA) method as described previously [39 (link)]. Briefly, an 80 µL of diluted fluorochrome stain (diluted first at 1:1000 in DMSO and followed by a 1:8 dilution in PBS) was added to the 20 µL of plasma sample or DNA standard solution in black 96-well plate (Cat# 655906, Greiner Bio-One, Frickenhausen, Germany). Fluorescent formation was recorded at 485 nm (excitation) and 535 nm (emission) by Synergy™ HTX Multi-Mode spectrofluorometer (BioTek Instruments, Winooski, VT, USA). The standard curve was established (0 to 5000 ng/mL) through serial dilution of UltraPure™ salmon sperm DNA (Cat# 15632011, Thermo Fisher Scientific, Waltham, MA, USA).
8
Generating Anti-dsDNA Antibody Immuno-Complexes
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ICs were prepared in vitro by incubation with Ultrapure salmon sperm DNA (Thermo Fisher Scientific) and sonicated into fragments ranging from 150 to 300 bp with anti-dsDNA antibodies engineered to have a human IgG1 backbone for 60 minutes in a 37°C water bath. The anti-dsDNA antibodies were provided by the autoimmunity, transplantation, and inflammation branch at Novartis, Switzerland.
9
Microalgae Transformation Protocols
10
Quantum Dot-Transcription Factor Binding Assay
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For a typical experiment, using a molar ratio of QD/TF/DNA = 1/4/18: 275 µL QDs at 0.15 µM in 1× HEPES with 1% BSA, were mixed with 275 µL SRTF1-his6 at 0.6 µM in 1× HEPES, at room temperature for 45 min. Double-stranded DNA labeled with a Cy5 fluorescent probe at the 3′ and 5′ ends (275 µL, 2.7 µM in 1× HEPES,) was added to the mixture. After 30 min, 220 µL of 1× HEPES, and 330 µL of 5× binding buffer (25 mM MgCl2, 25% glycerol, and 250 mg L−1 Invitrogen™ UltraPure™ Salmon Sperm DNA in 0.1 M Tris-HCl) were added and the mixture incubated for 15 min at RT.
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