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Experimental Protocols for Generation and Evaluation of Articular Cartilage
Published in Kyriacos A. Athanasiou, Eric M. Darling, Grayson D. DuRaine, Jerry C. Hu, A. Hari Reddi, Articular Cartilage, 2017
Kyriacos A. Athanasiou, Eric M. Darling, Grayson D. DuRaine, Jerry C. Hu, A. Hari Reddi
Reagents PicoGreen fluorescent dye1× Tris-EDTA (TE) bufferDNA standard (commonly lambda phage DNA)
Investigation of DNA Methylation in Autosomal Dominant Polycystic Kidney Disease
Published in Jinghua Hu, Yong Yu, Polycystic Kidney Disease, 2019
WGBS is the most comprehensive of all existing methods, permitting the genome-wide evaluation of DNA methylation at single-base resolution.68,79 For this reason, it is considered the “gold standard” for profiling DNA methylation.68 The most considerable limitations are the cost and difficulties in analysis of the next generation sequencing (NGS) data.67 Until recently, another limitation of WGBS was the considerable amount of DNA required for the analysis. However, there exist modified protocols capable of postponing the adaptor ligation step until after bisulfite treatment. This allows WGBS to be performed routinely from ∼30 ng or even as little as 125 pg of DNA.80 Below is a brief description of an established protocol adapted from studies in tissues.68Genomic DNA is isolated from kidney tissues and renal cells, using the MasterPure DNA Purification Kit (Epicentre), or comparable method, according to the manufacturer's instructions. This kit employs a nonenzymatic approach to cell lysis, followed by protein precipitation and subsequent nucleic acid isolation.Extracted DNA is resuspended in TE buffer (1× buffer: 10 mM Tris-HCl, 1 mM EDTA at pH 8.0) and quantified by fluorometry.Genomic DNA is converted by bisulfite treatment. Briefly, 50–100 ng of purified genomic DNA is treated with Zymo Lightning Conversion Reagent in a thermal cycle for 8 min at 98°C, followed by 60 min at 54°C. Note: Bisulfite treatment is known to fragment DNA.Bisulfite-treated DNA is purified on a spin column, and used to prepare a sequencing library using the EpiGnome Kit (Epicentre). In this procedure, bisulfite-treated single-stranded DNA is randomly primed using a polymerase able to read uracil nucleotides to synthesize DNA containing a specific sequence tag.The generated libraries are diluted and loaded onto the cBot DNA Cluster Generation System. After cluster generation is complete, the flow cell is transferred to the HiSeq 2500 System for sequencing using 75 bp paired-end reads.
HLA-DR and -DQ Typing by DNA-RFLP Analysis
Published in M. Kam, Jeffrey L. Bidwell, Handbook of HLA TYPING TECHNIQUES, 2020
DNA may be isolated from any nucleated cells, including those of poor physiological status. The rapid "salting-out" procedure22 is recommended. This method has proved the best for routine use because it avoids organic extraction of proteins, is both rapid and economical, and provides DNA of sufficient quality for restriction endonuclease digestion. Collect blood into sodium citrate or dipotassium EDTA anticoagulant. Avoid the use of heparin as an anticoagulant.Centrifuge 10 ml blood for 10 min at 1300 × g.Transfer buffy layer (approximately 1 ml) to a 10 ml disposal plastic conical tube. Add 8 ml red cell lysis buffer (0.144 M ammonium chloride, 1 mM sodium hydrogen carbonate), mix, then let stand for 20 min at room temperature.Centrifuge for 10 min at 1300 × g. Remove red cell lysate as near to the white cell pellet as possible. Resuspend the white cell pellet in 3 ml nuclei lysis buffer (10 mM Tris-HCl, pH 8.2, 0.4 M NaCl, 2 mM EDTA pH 8.0). Add 0.6 ml 1x Proteinase K solution (2 mg/ml protease K in 1% w/v sodium dodecyl sulfate (SDS), 2 mM EDTA pH 8.0.5x Proteinase K solution contains 10 mg/ml protease K in 1 % w/v SDS, 2 mM EDTA pH 8.0), and 0.2 ml 10% w/v SDS. Mix well.Incubate for 18 h in a 37°C water bath. (Note: If more convenient, 5x Protease K solution may be substituted in place of lx; in this case, incubate for 3 h at 55°C.)Add 1 ml 6 M NaCl, shake vigorously for 15 s.Centrifuge for 25 min at 1300 to 1500 × g.Avoiding the pellet, pipette the supernatant into a clean, disposable plastic conical tube. Add 8 ml absolute ethanol. Cap the tube and mix gently by inversion.Remove the precipitated DNA with a sealed glass Pasteur pipette, squeeze out the excess ethanol, and redissolve the DNA in 0.1 to 0.3 ml TE buffer. Store at 4°C until required.For assay, add 5 μl of the DNA solution to 495 μl of TE buffer and measure the optical density (OD) at 260 nm. The concentration (μg/μl) of DNA is 5 times the OD value.
Upregulation of antimicrobial peptide expression in slc26a3-/- mice with colonic dysbiosis and barrier defect
Published in Gut Microbes, 2022
Archana Kini, Bei Zhao, Marijana Basic, Urmi Roy, Aida Iljazovic, Ivan Odak, Zhenghao Ye, Brigitte Riederer, Gabriella Di Stefano, Dorothee Römermann, Christian Koenecke, André Bleich, Till Strowig, Ursula Seidler
Stool/fecal pellets were collected from gavaged mice and donor mice post sacrifice and immediately stored at −80°C. Stool pellets were similarly collected from the slc26a3 wt and ko mice at multiple time points (Weeks 5, 8, 12, 16 and 20 for colon and Early: 4–9 weeks, Mid: 10–15 weeks and Late: 16–20 weeks for cecum) and stored at −80°C. DNA was extracted using a method as described previously.79 Briefly, samples were first suspended in a solution containing 200 µL 0.1 mm zirconia/silica beads, 500 µL extraction buffer (200 mM NaCl, 200 mM Tris [pH 8.0] and 20 mM Sodium EDTA), 200 µL 20% SDS and 500 µL phenol:chloroform:isoamyl alcohol mixture (pH 7.9, 25:24:1). The samples were subsequently lysed twice by mechanical disruption using bead beater for 2 min. It was then followed by extraction with phenol:chloroform:isoamyl alcohol (pH 7.9, 25:24:1), and precipitation with ice-cold isopropanol. The DNA extracts were resuspended in Tris-EDTA (TE) buffer.
Lactobacillus casei reduces the extracellular matrix components of fluconazole-susceptible Candida albicans biofilms
Published in Biofouling, 2021
Beatriz H. D. Panariello, Marlise Inez Klein, Luana Mendonça Dias, Amanda Bellini, Vitoria Bonan Costa, Paula Aboud Barbugli, Ana Claudia Pavarina
For eDNA analysis, a 0.65 ml aliquot of phenol: chloroform: isoamyl alcohol (25: 24: 1) was mixed with 0.65 ml of the biofilm supernatant and then, after centrifugation (10000 × g, 5 min), the aqueous phase was transferred to a new tube and mixed with chloroform: isoamyl alcohol (24:1). After centrifugation (10,000 × g, 5 min), the aqueous phase was transferred again to a new tube, mixed with three volumes of isopropanol and 1/10 volume of 3 M sodium acetate and stored for precipitation (-20 °C; 18 h). After precipitation, the tubes were centrifuged (13,000 × g; 20 min; 4 °C) and washed three times with cold 70% ethanol. After that, the samples were dried and 10 µl of TE buffer (1 mM Tris HCl/EDTA, pH 8.0) were added to each sample. The determination of eDNA was performed at OD 260 nm.
Clonal evolution of Candida albicans, Candida glabrata and Candida dubliniensis at oral niche level in health and disease
Published in Journal of Oral Microbiology, 2021
Alexander J. Moorhouse, Rosa Moreno-Lopez, Neil A.R. Gow, Karolin Hijazi
Pelleted overnight liquid YPD cultures were transferred to 2 mL Eppendorf tubes and vortexted for 5 min in the presence of 0.3 g of acid washed glass beads, 200 μL extraction buffer (2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris pH 8.0, 1 mM EDTA), 200 μL phenol: chloroform alcohol (1:1). TE buffer (200 μL, Tris EDTA pH 7.5) was added and the tubes inverted. Phase separation by centrifugation for 5 min at 14,000 g was followed by transfer of the aqueous layer to 1 mL of 100% EtOH in 2 mL tubes and centrifugation for 2 min at 14,000 g to obtain precipitated DNA. Pelleted DNA was resuspended in 400 μL TE plus 10 μL RNase A (10 mg ml-1) and incubated for 20 min at 37°C, subsequent precipitation in 20 μL 3 M NaOAc and 1 mL 100% EtOH with centrifugation at 14,000 g for 10 min was followed by resuspension in 200 μL sterile H2O, as previously described [40].