The ucfDNA was extracted from 2–3 mL of the centrifuged urine using the Wizard Plus Minipreps DNA Purification System (Promega, Madison, Wisconsin). Each 1 mL of urine was mixed with 1.5 mL of 6 M guanidine (Sigma–Aldrich, St. Louis, Missouri). The mixture was incubated with 1 mL of resin for 2 h at room temperature with gentle mixing. The resin-DNA complex was then purified using the Wizard mini-column. The extracted ucfDNA sample was eluted with 70 μL nuclease-free water after centrifugation at 10,000 g for 1 min. Those samples with quantifiable ucfDNA were used for the downstream sequencing library preparation. A total of 44 pooled urine samples had quantifiable ucfDNA by the Qubit dsDNA high sensitivity assay with a Qubit Fluorometer (ThermoFisher Scientific), consisting of 18, 5, and 21 samples from wildtype, Dnase1l3-/-, and Dnase1-/- groups, respectively.
6 m guanidine
Manufactured by Merck Group
6 M guanidine is a chemical reagent commonly used in molecular biology and biochemistry laboratories. It is a concentrated solution of guanidine hydrochloride. Guanidine is a denaturing agent that can be used to solubilize and disrupt proteins, DNA, and other biomolecules. This product is often utilized in various laboratory procedures, such as protein purification, cell lysis, and nucleic acid extraction and purification.
Automatically generated - may contain errors
Lab products found in correlation
Qubit fluorometer, by Thermo Fisher Scientific (1 mentions)
Wizard plus minipreps dna purification system, by Promega (1 mentions)
Dithiothreitol (dtt), by Merck Group (1 mentions)
Ltq xl mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Dionex ultimate 3000, by Thermo Fisher Scientific (1 mentions)
Trypsin, by Promega (1 mentions)
Ab 8gx, by Merck Group (1 mentions)
3 protocols using 6 m guanidine
1
Urine-Derived Cell-Free DNA Purification
2
Quantitative Analysis of Glycosylation in IVIG/Fc
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IVIG/Fc at 100 mg/mL was diluted into 6 M Guanidine (Sigma) to a final concentration of 2 mg/mL. Dithiothreitol was added to a concentration of 10 mM, and the protein was denatured by reducing the disulfide bonds at 65 °C for 30 min. After cooling on ice, the samples were incubated with 30 mM iodoacetamide for 1 h in the dark to carbamidomethylate the cysteine residues. Guanidine, Dithiothreitol, and iodoacetamide were all purchased from Sigma. The protein was then dialyzed across a 10-kDa membrane into 25 mM (pH 7.8) ammonium bicarbonate buffer. Trypsin (Promega, Madison, WI) was added to the sample for proteolysis, which was carried out in a Barocycler (NEP 2320; Pressure Biosciences, South Easton, MA). The pressure was cycled between 20 kpsi and ambient pressure at 37 °C for a total of 30 cycles in 1 h.
LC-MS/MS analysis of the tryptic digests was performed on a Dionex Ultimate 3000 chromatography system and a LTQ-XL mass spectrometer (Thermo Fisher Scientific, San Jose, CA). Peptides were separated on a Waters BEH PepMap column (Waters, Milford, MA) using 0.1% formic acid in water and 0.1% formic acid in acetonitrile as the mobile phases.
Glycosylation was quantified for the different isotypes by the area from the extracted ion chromatogram (XIC) for the tryptic glycopeptides as described by Zauner et al.20 (link)
LC-MS/MS analysis of the tryptic digests was performed on a Dionex Ultimate 3000 chromatography system and a LTQ-XL mass spectrometer (Thermo Fisher Scientific, San Jose, CA). Peptides were separated on a Waters BEH PepMap column (Waters, Milford, MA) using 0.1% formic acid in water and 0.1% formic acid in acetonitrile as the mobile phases.
Glycosylation was quantified for the different isotypes by the area from the extracted ion chromatogram (XIC) for the tryptic glycopeptides as described by Zauner et al.20 (link)
3
Alcian Blue Staining of ATDC5 Micromasses
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ATDC5 micromasses were washed with PBS and fixed with 95% ice-cold methanol for 30 min at 4 C. After rinsing three times with water, micromasses were stained with Alcian Blue (AB) (0.1% AB 8GX, Sigma), washed three times with water and air dried. Quantification of the staining was performed by dissolving the micromasses with 6 M guanidine (Sigma) and measuring the absorbance at 595 nm with a spectrophotometer (BioTek Synergy).
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