U-TRINZOL Reagent 100 ml - UniscienceSKU: UNI-R3152
Overview U-TRINZOL reagent is a ready-to-use monophasic solution of phenol and guanidine isothiocyanate for total RNA isolation from cells and tissues. During sample homogenization or lysis, U-TRINZOL Reagent maintains the integrity of RNA while disrupting cells and dissolving cell components. Addition of chloroform followed by centrifugation, separates the solution into an aqueous phase (RNA) and an organic phase (DNA and Proteins). After transfer of the aqueous phase, RNA is recovered by precipitation with isopropyl alcohol. DNA and proteins can be recovered by sequential precipitation with ethanol which yields DNA from the interphase, and an additional precipitation with isopropyl alcohol which yields Proteins from the organic phase. Co-purification of DNA may be useful for normalizing RNA yields from sample to sample. This technique performs well with small and large quantities of tissue (50-10 mg) and cells (5 x 10 – 10 ) of human, animal, plant or bacterial origin. The simplicity of the U-TRINZOL Reagent method allows simultaneous processing of a large number of samples within one hour. Total RNA isolated by U-TRINZOL Reagent is free of protein and DNA contamination. APPLICATIONS Total RNA isolated by U-TRINZOL can be used for Northern Blot analysis, dot blot hybridizations, poly (A)+ selection, in vitro translation, RNase protection assay and molecular cloning. If the RNA is to be used for RT PCR, it should be treated with molecular biology grade DNase I. SIZE AND STORAGE U-TRINZOL reagent, when stored in indicated temperature is stable for 12 months. Reagents required, but not supplied: 1. Chloroform 2. Isopropyl alcohol 3. 75% Ethanol (in RNA-free water) 4. RNase-free water or 0.5% SDS solution WARNING: Toxic in contact with skin and if swallowed causes burns. After contact with skin, wash immediately with plenty of detergent and water. If you feel unwell, seek medical advice (show label when possible). Phenol (108-95-2) and other Components (NJTSRN 80100437-5000p). PRECAUTIONS FOR PREVENTING RNASE CONTAMINATION The following guidelines should be conducted when working with RNA. 1. Always wear disposable gloves. 2. Use sterile, disposable plasticware and automatic pipettes reserved for RNA work to prevent cross-contamination with RNases from shared equipment. 3. In the presence of U-TRINZOL Reagent, RNA is protected from RNase contamination. Downstream sample handling requires nondisposable glassware or plasticware to be RNase-free. Glass items can be baked at 150°C for 4 hours and plastic items can be soaked for 10 minutes in 0.5 M NaOH, rinsed thoroughly with water and autoclaved. RECOMMENDED U-TRINZOL VOLUME ON DIFFERENT STARTING MATERIALS 1. HOMOGENIZATION Use U-TRINZOL Reagent to prepare lysates from various sample types as described below. 1 a. TISSUES: From animal or plant (either fresh or frozen at -70°C until use) can be processed by freezing with liquid nitrogen and grinding into a fine powder using a mortar and pestle. Homogenize tissue samples in 1 ml U-TRINZOL Reagent per 50–100 mg tissue using a tissue homogenizer or rotor-stator. 1b. ADHERENT CELLS: Lyse cells directly in a culture dish by adding 1 ml of U-TRINZOL Reagent to a 3.5 cm diameter dish and passing the cell lysate several times through a pipette. The amount of U-TRINZOL Reagent added is based on the area of the culture dish (1 ml per 10 cm ) and not on the number of cells present. An insufficient amount of U-TRINZOL Reagent may result in contamination of the isolated RNA with DNA. 1c. SUSPENSION CELLS: Pellet cells by centrifugation. Lyse cells in U-TRINZOL Reagent by repetitive pipetting. Use 1 ml of the reagent per 5-10 × 10 of animal, plant or yeast cells, or per 1 × 10 bacterial cells. Washing cells before addition of U-TRINZOL Reagent should be avoided as this increases the possibility of mRNA degradation. Disruption of some yeast and bacterial cells may require the use of a homogenizer. OPTIONAL: An additional isolation step may be required for samples with high content of proteins, fat, polysaccharides or extracellular material such as: muscles, fat tissue, and tuberous parts of plants. Following homogenization, remove insoluble material from the homogenate by centrifugation at 12,000 × g for 10 minutes at 2 to 8°C. The resulting pellet contains extracellular membranes, polysaccharides, and high molecular weight DNA, while the supernatant contains RNA. In samples from fat tissue, an excess of fat collects as a top layer which should be removed. In each case, transfer the cleared homogenate solution to a fresh tube and proceed with chloroform addition and phase separation as described. 2. PHASE SEPARATION Incubate the homogenized samples for 5 minutes at 15 to 30°C to permit the complete dissociation of nucleoprotein complexes. Add 0.2 ml of chloroform per 1 ml of U-TRINZOL Reagent. Cap sample tubes securely. Shake tubes vigorously by hand for 15 seconds and incubate them at room temperature for 2 to 3 minutes. Centrifuge the samples at 12,000 × g for 5-10 minutes at 4°C. Following centrifugation, the mixture separates into a lower yellow, phenol-chloroform phase, an interphase, and a colorless upper aqueous phase. RNA remains exclusively in the aqueous phase. The volume of the aqueous phase is about 60% of the volume of U-TRINZOL Reagent used for homogenization. 3. RNA PRECIPITATION Transfer the aqueous phase to a fresh tube, and save the organic phase if isolation of DNA or protein is desired. Precipitate the RNA from the aqueous phase by mixing with isopropyl alcohol. Use 0.5 ml of isopropyl alcohol per 1 ml of U-TRINZOL Reagent used for the initial homogenization. Mix well by inverting the tube several times. Incubate samples at 15 to 30°C for 10 minutes and centrifuge at 12,000 × g for 5-10 minutes at 4°C. The RNA precipitate, often invisible before centrifugation, forms a gel-like pellet on the side and bottom of the tube.