Reagents and buffers used for the silica column method (adapted method from Cremonesi et al., 2006 using the silica column) and boil on silica column method (adapted Barea et al., 2004) , were prepared based on the protocol described by Cremonesi et al. (2006) . First, 0.5 M EDTA, pH 8.0, 100 mM Tris-HCl, pH 6.8, and 1 M Tris-HCl, pH 8.0, solutions were prepared. Next, lysis buffer (AL buffer) was prepared by adding 2 g Triton, 5 mL 100 mM Tris-HCl, pH 6.8, 2 mL 0.5 M EDTA, 17.724 g guanidine thiocyanate (Sigma-Aldrich, St. Louis, MO, USA), 0.5 g DL.T, and 50 mL QSP MilliQ water. The washing buffers 1 and 2 (AW1 and AW2 buffers) were prepared by adding 0.146 g NaCl, 6.25 mL absolute ethanol, 6.25 mL isopropanol, 8.862 g guanidine thiocyanate (Sigma-Aldrich), 250 μL 1 M Tris-HCl, pH 8, and 35 mL QSP MilliQ water. However, guanidine hydrochloride (Sigma-Aldrich) was not added to wash buffer 2 (AW2 buffer); instead, 25 mL QSP MilliQ water was used. The elution buffer (AE buffer) was prepared by adding 150 μL 1 M Tris-HCl, pH 8.0, 30 μL 0.5 M EDTA, and 15 mL QSP MiliQ water.
Guanidine thiocyanate
Manufactured by Merck Group
Sourced in United States, Germany
Guanidine thiocyanate is a laboratory reagent used as a chaotropic agent in various applications, such as in the extraction and purification of nucleic acids. It disrupts hydrogen bonds and denatures proteins, enabling the effective isolation of DNA and RNA from biological samples.
Automatically generated - may contain errors
Lab products found in correlation
N lauroylsarcosine, by Merck Group (4 mentions)
Bathocuproine bcp, by Xi'an Yuri Solar Co. (3 mentions)
Fastdna spin kit for soil, by MP Biomedicals (2 mentions)
Fastprep 24, by MP Biomedicals (2 mentions)
Zirconia silica bead, by Biospec (2 mentions)
Proteinase k, by Merck Group (2 mentions)
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Hiseq 1500, by Illumina (1 mentions)
Wizard dna clean up system, by Promega (1 mentions)
Sybr green master mix, by Bio-Rad (1 mentions)
Bathocuproine, by Merck Group (1 mentions)
Lead iodide pbi2, by Tokyo Chemical Industry (1 mentions)
Tin 2 fluoride, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
N dimethylformamide, by Merck Group (1 mentions)
Ethylenediamine, by Merck Group (1 mentions)
Cesium iodide, by Merck Group (1 mentions)
Formamidinium iodide, by Greatcell Solar (1 mentions)
Methylammonium iodide, by Greatcell Solar (1 mentions)
Fluorescein, by Bio-Rad (1 mentions)
30 protocols using guanidine thiocyanate
1
Silica Column DNA Extraction Protocol
2
Illumina Sequencing of Bacteriophage CRB2
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The genome sequencing of CRB2 was carried out at a commercial local facility (INDEAR, Instituto de Agro-Biotecnología de Rosario, Rosario, Argentina) using Illumina HiSeq 1500 technology. Libraries were generated by using the Nextera1XT DNA Sample Preparation Guide Illumina (October 2012, Illumina Inc, San Diego, CA, USA). To obtain the template bacteriophage DNA high-titer bacteriophage lysates (generated on plates using 0.4% (w/v) agarose top medium) were treated as described previously with DNAse and RNAse treatment and filtration through 0.2 μm cellulose acetate filters followed by virion disruption by the addition of guanidine thiocyanate (Sigma, final concentration 800 mg/mL). After this step, was added to the cleared lysates and the mixture was gently mixed at room temperature for 2 h for full solubilization of this salt. Bacteriophage DNA was extracted from this suspension by using Wizard DNA Clean-Up System (Promega) according to the manufacturer instructions. Quantitation of bacteriophage DNA was estimated at Abs260 nm and DNA integrity was assessed by agarose gel electrophoresis. DNA was finally stored at -20°C until further use.
3
Quantitative Real-Time PCR Protocols
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Regular, real-time PCR was performed as described.58 (link) Briefly, total RNA was extracted from tissues or cells using a kit from Geneaid TriRNA isolation kit or TRIzol. 1 μg RNA was used for reverse transcription and real-time PCR using iScript RT kits and SYBR green master mix (Bio-Rad). Primers for U6 or GAPDH were used as internal controls for normalization. The sequences of primers are listed in Table S1.
Single-cell real-time PCR was performed as described.59 (link) In brief, the cultured cells were trypsinized and suspended in basal medium. A single cell was collected with a glass capillary under an avert phase contrast microscope. After being washed with PBS, the picked cell was lysated with 2 μL lysis buffer (containing 50 mM guanidine thiocyanate, Sigma-Aldrich). All the lysated samples were used to synthesize cDNA with a RT kit in 10 μL. Real-time PCR was performed with a miScriptSYBR GreenPCR Kit (Qigen) using 2 μL cDNA as template. For in vivo single-cell real-time PCR, the tumor tissues were washed with PBS, cut into small pieces, and cultured in the basal medium for 12 h. The attached tumor cells were trypsinized and suspended in basal medium. A single cell was collected, lysed, and processed to real-time PCR as above.
Single-cell real-time PCR was performed as described.59 (link) In brief, the cultured cells were trypsinized and suspended in basal medium. A single cell was collected with a glass capillary under an avert phase contrast microscope. After being washed with PBS, the picked cell was lysated with 2 μL lysis buffer (containing 50 mM guanidine thiocyanate, Sigma-Aldrich). All the lysated samples were used to synthesize cDNA with a RT kit in 10 μL. Real-time PCR was performed with a miScriptSYBR GreenPCR Kit (Qigen) using 2 μL cDNA as template. For in vivo single-cell real-time PCR, the tumor tissues were washed with PBS, cut into small pieces, and cultured in the basal medium for 12 h. The attached tumor cells were trypsinized and suspended in basal medium. A single cell was collected, lysed, and processed to real-time PCR as above.
4
Fractionation of Urine DNA
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Freshly collected urine was immediately mixed with 0.5 mol/L EDTA, pH 8.0, to a final concentration of 10 mmol/L EDTA and stored at − 20 °C. Total urine DNA was isolated by adding an equal volume of 6 mol/L guanidine thiocyanate (Sigma, St. Louis, MO) to thawed urine as described previously [17 (link)]. The fractionated high molecular weight (HMW) urine DNA (> 1 kb) and low molecular weight (LMW) urine DNA (≤1 kb) was obtained from total urine DNA using carboxylated magnetic beads (Agentcourt Bioscience Corporation, Beverly, MA), as previously described [22 (link)].
5
Perovskite Solar Cell Fabrication
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Patterned glass-based ITO was purchased from Shenzhen Huayu Union Technology Co., Ltd. Poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonate) (PEDOT: PSS) was purchased from Heraeus Precious Metals GmbH. Tin iodide (SnI2), Cesium iodide (CsI), bathocuproine (BCP), tin powder (Sn), N, N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), toluene, tin (II) fluoride (SnF2), guanidine thiocyanate (GuaSCN), and ethylenediamine (EDA) were purchased from Sigma-Aldrich. Methylammonium iodide (MAI) and formamidinium iodide (FAI) were purchased from Greatcell Solar Materials. Lead iodide (PbI2) and [2-(9H-carbazol-9-yl)ethyl]phosphonic acid (2PACz) were purchased from Tokyo Chemical Industry Co. Ltd (TCI). C60 was purchased from Puyang Yongxin Fullerene Technology Co., Ltd. Quartz-based ITO was purchased from Luoyang Guluo Glass Co. Ltd. All materials and solvents were used as received without any further purification.
6
Molecular Techniques in Genomic Research
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BNF, SYBR® Green I (10,000× concentration), agarose, JumpStart Taq DNA polymerase, Enhanced Avian RT first-strand synthesis kit (STR-1), GenElute™ PCR Clean-Up Kit, GenElute™ HP Endotoxin-Free Plasmid Maxiprep Kit, PCR Low Ladder Marker Set, guanidine thiocyanate, ammonium thiocyanate, Williams’ E medium, LB broth, and LB agar were supplied by Sigma-Aldrich Co (St. Louis, MO, USA). Fluorescein was obtained from Bio-Rad Laboratories (Hercules, CA, USA). Restriction endonucleases were purchased from Fermentas International Inc. (Burlington, Canada). Deoxyribonucleotide triphosphates such as dATP, dGTP, aCTP, and dTTP were provided by Roche Diagnostics (Mannheim, Germany). PCR primers were provided by Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Poland (oligo.pl), and Genomed, Poland. Agilent RNA 6000 Reagents were provided by Agilent Technologies (Santa Clara, CA, USA). Affymetrix Human Genome U219 Array Strip, GeneChip 3′IVT Express Kit and GeneAtlas Hybridization, and Wash and Stain Kit for 3′IVT Arrays were provided by Affymetrix (Santa Clara, CA, USA). GeneClip™ U1 Hairpin Cloning System—Neomycin Vector and antibiotic G418 (Geneticin)—was provided by Promega (Madison, WI, USA). Lipofectamine 2000 and Opti-MEM® I Reduced Serum Medium was provided by Invitrogen (Carlsbad, CA, USA). All the other compounds were readily available as commercial products.
7
Biogas Reactor Sampling and DNA Extraction
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Samples were taken on five different time-points (date: 150303, 150414, 150519, 150709, and 151117) from a continuous stirred-tank biogas reactor (GR2) in the Anaerobic Microbiology and Biotechnology Laboratory, Swedish University of Agricultural Sciences, Uppsala. The reactor was operated with mixed food waste at 37°C, an organic loading rate of 2.5 ± 0.42 g VS L–1 day–1 and NH4+-N 5.4 g/L, while other operating parameters were as described for reactor DTE37 by Westerholm et al. (2015) (link). Genomic DNA extraction was performed with the FastDNA™ Spin kit for soil (MP Biomedicals, 2015 ) with an additional wash step with 500 μL 5.5 M Guanidine thiocyanate (Sigma-Aldrich, 2020 ) for humic acid removal (Singh, 2020 (link)). Reactor and DNA samples were stored at −20°C until further processing.
8
Metagenomic DNA Extraction from Murine Cecal Content
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Metagenomic DNA was obtained from cecal content of fasted SPF mice after mechanical lysis followed by purification according to a published protocol [60 (link)] modified as follows: cecal content in 600 μl stool DNA stabilizer (Stratec Biomedical AG) was transferred into a 2-ml screw-cap tube containing 500 mg zirconia/silica beads (0.1 mm; BioSpec Products), 250 μl 4 M Guanidinethiocyanate (Sigma-Aldrich, Germany), and 500 μl 5% N-lauroylsarcosine (Sigma-Aldrich, Germany). Samples were mixed and incubated for 60 min at 70 °C with constant shaking, and bacterial cells were disrupted by mechanical lysis using a FastPrep®-24 (three times, 40 s, 6.5 m/sec) (MP Biomedicals) fitted with a cooling adaptor. After addition of 15 mg polyvinylpolypyrrolidone (PVPP, Sigma-Aldrich, Germany), the suspension was vortexed and centrifuged (3 min, 15,000×g, 4 °C). The supernatant (500 μl) was transferred into a new Eppendorf tube, mixed with 5 μl RNase (VWR International, stock concentration 10 mg/ml) and incubated for 20 min at 37 °C with constant shaking. Genomic DNA was purified using NucleoSpin® gDNA columns (Macherey Nagel GmbH & Co. KG, Germany) following the manufacturer’s instructions. DNA quantity and quality were measured with a NanoDrop® instrument (Thermo Fisher Scientific Inc., Germany).
9
Urine DNA Extraction and Bisulfite Conversion
10
Urinary DNA Extraction for Kidney Cancer
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Urine samples of kidney cancer were recruited from the Department of Surgery of the Prince of Wales Hospital, Hong Kong. Healthy samples were recruited from the Department of Chemical Pathology of the Prince of Wales Hospital, Hong Kong. All subjects involved in this study gave written informed consent, and the study was approved by The Joint Chinese University of Hong Kong–Hospital Authority New Territories East Cluster Clinical Research Ethics Committee, under the Declaration of Helsinki. Collection and storage of fresh urine were followed as previously described17 (link). Urinary DNA was extracted from 10 mL–30 mL cell-free urine component with the Wizard Plus Minipreps DNA Purification System; (Promega). 15 mL of 6 mol/L guanidine thiocyanate (Sigma–Aldrich) and 1 mL of resin (Wizard Plus Minipreps DNA Purification System; Promega) were added in each 10 mL of the processed urine. The mixture was incubated and shaken gently at room temperature for 2 h. Then, the DNA was purified and eluted into 100 μL RNase-free water using the minicolumns and according to the manufacturer’s instruction of the purification system. The quantity of urinary DNA obtained ranged from ~10 ng to ~100 ng according to Qubit assays.
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