TRIzol® Reagent is a complete, ready-to-use reagent for the isolation of high-quality total RNA or the simultaneous isolation of RNA, DNA, and protein from a variety.
TRIzol™ Reagent, TRIzol™ LS Reagent, TRIzol™ Invitrogen, TRIZOL REAGENT 100ML, IPrep™ Trizol™ Plus RNA Kit 52 preps, TRIzol™ Max™ Bacterial RNA.
TRIzol « Reagent
Invitrogen Gateway Cloning Manual Pdf to Invitrogens. multisite gateway cloning manual file type. was extracted using TRIzol reagent (Invitrogen) and purified using.
Caution Determining Yield of Protein Troubleshooting Contents and Storage Cause Problem Low yield of RNA, Low yield of DNA, or Low yield of protein Intended Use RNA is degraded, DNA is degraded, or Protein is degraded. Materials Needed RNA contamination or DNA contamination Protein Isolation DNA Isolation RNA Isolation Low A260/280 for RNA Low A260/280 for DNA References: Preparing Samples Homogenizing samples DNA Isolation Procedure Note: The mixture separates into a lower red phenol-chloroform phase, an interphase, and a colorless upper aqueous phase. RNA remains exclusively in the aqueous phase. The upper aqueous phase is ~50% of the total volume. Determining Yield of RNA and DNA RNA Isolation Procedure Expected yields Protein Isolation Procedure 15596026.PPS_Trizol_Reagent_13Dec2012[1].pdf TRIzol« Reagent Catalog Numbers 15596-026 15596-018 LearnáMoreá Quantity 100 mL 200 mL Buy Now Buy Now Store at 2░C to 25░C Description TRIzol« Reagent is a ready-to-use reagent, designed to isolate high quality total RNA (as well as DNA and proteins) from cell and tissue samples of human, animal, plant, yeast, or bacterial origin, within one hour. TRIzol« Reagent is a monophasic solution of phenol, guanidine isothiocyanate, and other proprietary components which facilitate the isolation of a variety of RNA species of large or small molecular size. TRIzol« Reagent maintains the integrity of the RNA due to highly effective inhibition of RNase activity while disrupting cells and dissolving cell components during sample homogenization. TRIzol« Reagent allows for simultaneous processing of a large number of samples, and is an improvement to the single-step RNA isolation method developed by Chomcynski and Sacchi (Chomczynski & Sacchi, 1987). TRIzol« Reagent allows the user to perform sequential precipitation of RNA, DNA, and proteins from a single sample (Chomczynski, 1993). After homogenizing the sample with TRIzol« Reagent, chloroform is added, and the homogenate is allowed to separate into a clear upper aqueous layer (containing RNA), an interphase, and a red lower organic layer (containing the DNA and proteins). RNA is precipitated from the aqueous layer with isopropanol. DNA is precipitated from the interphase/organic layer with ethanol. Protein is precipitated from the phenol-ethanol supernatant by isopropanol precipitation. The precipitated RNA, DNA, or protein is washed to remove impurities, and then resuspended for use in downstream applications. • • • Isolated RNA can be used in RT-PCR, Northern Blot analysis, Dot Blot hybridization, poly(A)+ selection, in vitro translation, RNase protection assay, and molecular cloning. Isolated DNA can be used in PCR, Restriction Enzyme digestion, and Southern Blots. Isolated protein can be used for Western Blots, recovery of some enzymatic activity, and some immunoprecipitation. Caution TRIzol« Reagent contains phenol (toxic and corrosive) and guanidine isothiocyanate (an irritant), and may be a health hazard if not handled properly. Always work with TRIzol« Reagent in a fume hood, and always wear a lab coat, gloves and safety glasses. Contact your Environmental Heath and Safety (EH&S) department for proper work and disposal guidelines. Avoid direct contact with TRIzol« Reagent, because contact to skin, eyes, or respiratory tract may cause chemical burns to the exposed area. If contact to skin or eyes occurs, immediately wash the exposed area with copious amounts of water for 15 minutes and seek medical attention if necessary. If you inhale vapors, move to fresh air and seek medical attention if necessary. For more information, refer to the TRIzol« Reagent SDS (Safety Data Sheet), available from our web site at www.lifetechnologies.com/support. Contents and Storage TRIzol« Reagent is supplied in 100 mL (Cat. no. 15596-026) or 200 mL (Cat. no. 15596-018) volumes, and shipped at room temperature. Upon receipt, store TRIzol« Reagent at room temperature. TRIzol« Reagent is stable for 12 months when properly stored. Intended Use For research use only. Not intended for human or animal diagnostic or therapeutic uses. Materials Needed The following additional materials are needed, but not supplied for the isolation of RNA, DNA or proteins. RNA Isolation • Chloroform • Isopropyl alcohol • 75% ethanol (in DEPC-treated water) • RNase-free water or 0.5% SDS • Centrifuge and rotor capable of reaching up to 12,000 Î g • Polypropylene microcentrifuge tubes • Water bath or heat block (55–60░C) DNA Isolation • • • • • • Chloroform 100% ethanol 75% ethanol 0.1 M sodium citrate in 10% ethanol 8 mM NaOH Centrifuge and rotor capable of reaching up to 12,000 Î g • Polypropylene microcentrifuge tubes Protein Isolation • • • • • • Chloroform Isopropyl alcohol 100% ethanol 0.3 M Guanidine hydrochloride in 95% ethanol 1% SDS Centrifuge and rotor capable of reaching up to 12,000 Î g • Polypropylene microcentrifuge tubes Part no. 15596026.PPS MAN0001271 Rev. Date: 13 Dec 2012 For support, visit www.lifetechnologies.com/support or email [email protected]. To reorder, visit www.lifetechnologies.com Preparing Samples Homogenizing samples 1. Determine your sample type, and perform homogenization at room temperature according to the table below. The sample volume should not exceed 10% of the volume of TRIzol« Reagent used for homogenization. Be sure to use the indicated amount of TRIzol« Reagent, because an insufficient volume can result in DNA contamination of isolated RNA. Sample Type Tissues Action 1. Add 1 mL TRIzol« Reagent per 50–100 mg of tissue sample. 2. Homogenize sample using a glassTeflon« or power homogenizer. Note: Process or freeze tissue samples immediately upon collection. 1. Remove growth media from culture dish. 2. Add 1 mL TRIzol« Reagent directly to the cells in the culture dish per 10 cm2 of culture dish surface area. Note: Add 1 mL TRIzol« Reagent for a 35 mm dish, 3 mL for a 60 mm dish, and 8 mL for a 100 mm dish. 3. Lyse the cells directly in the culture dish by pipetting the cells up and down several times. 1. 2. Harvest cells by centrifugation and remove media. Add 0.75 mL of TRIzol« Reagent per 0.25 mL of sample (5–10 Î 106 cells from animal, plant or yeast origin, or 1 Î 107 cells of bacterial origin). Note: Do not wash cells before addition of TRIzol« Reagent to avoid increased chance of mRNA degradation. Lyse cells in sample by pipetting up and down several times. Disruption of some yeast and bacterial cells may require the use of a homogenizer. Phase separation 1. Incubate the homogenized sample (see Homogenizing samples) for 5 minutes at room temperature to permit complete dissociation of the nucleoprotein complex. Add 0.2 mL of chloroform per 1 mL of TRIzol« Reagent used for homogenization. Cap the tube securely. Shake tube vigorously by hand for 15 seconds. Incubate for 2–3 minutes at room temperature. Centrifuge the sample at 12,000 Î g for 15 minutes at 4░C. Note: The mixture separates into a lower red phenolchloroform phase, an interphase, and a colorless upper aqueous phase. RNA remains exclusively in the aqueous phase. The upper aqueous phase is ~50% of the total volume. Remove the aqueous phase of the sample by angling the tube at 45░ and pipetting the solution out. Avoid drawing any of the interphase or organic layer into the pipette when removing the aqueous phase. Place the aqueous phase into a new tube and proceed to the RNA Isolation Procedure. Save the interphase and organic phenol-chloroform phase if isolation of DNA or protein is desired. See DNA Isolation Procedure and Protein Isolation Procedure for details. The organic phase can be stored at 4░C overnight. 2. 3. 4. 5. Adherent Cells (Monolayer) 6. 7. 8. Suspension Cells RNA Isolation Procedure Always use the appropriate precautions to avoid RNase contamination when preparing and handling RNA. RNA precipitation 1. (Optional) When precipitating RNA from small sample quantities (<106 cells or <10 mg tissue), add 5–10 Ág of RNase-free glycogen as a carrier to the aqueous phase. Note: Glycogen is co-precipitated with the RNA, but does not inhibit first-strand synthesis at concentrations ≤4 mg/mL, and does not inhibit PCR. Add 0.5 mL of 100% isopropanol to the aqueous phase, per 1 mL of TRIzol« Reagent used for homogenization. Incubate at room temperature for 10 minutes. Centrifuge at 12,000 Î g for 10 minutes at 4░C. Note: The RNA is often invisible prior to centrifugation, and forms a gel-like pellet on the side and bottom of the tube. Proceed to RNA wash. Remove the supernatant from the tube, leaving only the RNA pellet. Wash the pellet, with 1 mL of 75% ethanol per 1 mL of TRIzol« Reagent used in the initial homogenization. Note: The RNA can be stored in 75% ethanol at least 1 year at –20░C, or at least 1 week at 4░C. Vortex the sample briefly, then centrifuge the tube at 7500 Î g for 5 minutes at 4░C. Discard the wash. Vacuum or air dry the RNA pellet for 5–10 minutes. Do not dry the pellet by vacuum centrifuge. Note: Do not allow the RNA to dry completely, because the pellet can lose solubility. Partially dissolved RNA samples have an A260/280 ratio <1.6. Proceed to RNA resuspension. 3. 2. (Optional) When preparing samples with high content of fat, proteins, polysaccharides, or extracellular material (e.g., muscle, fat tissue, or tuberous plant material), an additional isolation step may be required to remove insoluble material from the samples. Note: Do not perform this additional isolation step if you are performing subsequent DNA isolation on your sample. Sample Type Tissue or cells with high content of fat, protein, polysaccharide, or extracellular material Notes 1. Following homogenization, centrifuge your sample at 12,000 Î g for 10 minutes at 4░C. Note: The resulting pellet contains ECM, polysaccharides, and high molecular weight DNA, while the supernatant contains the RNA. In high fat content samples, a layer of fat collects above the supernatant. 2. Remove and discard the fatty layer. 3. Transfer the cleared supernatant to a new tube. 2. 3. 4. 5. 1. 2. RNA wash 3. 4. 3. Proceed to Phase separation, or store the homogenized sample. Homogenized samples can be stored at room temperature for several hours, or at –60 to –70░C for at least one month. 5. RNA resuspension 1. Resuspend the RNA pellet in RNase-free water or 0.5% SDS solution (20–50 μL) by passing the solution up and down several times through a pipette tip. Note: Do not dissolve the RNA in 0.5% SDS if it is to be used in subsequent enzymatic reactions. Incubate in a water bath or heat block set at 55–60░C for 10–15 minutes. Proceed to downstream application, or store at –70░C. DNA resuspension Resuspend the DNA in 8mM NaOH at a concentration of 0.2–0.3 Ág/ÁL. 1. Add 0.3–0.6 mL of 8mM NaOH per 50–70 mg of tissue, or per 1 Î 107 cells. Note: Resuspending the DNA is a mild base is highly recommended because isolated DNA does not resuspend well in water or Tris buffer. 2. Remove any insoluble material by centrifuging the sample at 12,000 Î g for 10 minutes at 4░C. 3. Transfer the supernatant containing the DNA to a new tube. Adjust pH as needed with HEPES and proceed to downstream application of choice. The DNA can be stored overnight at 4░C, but for long-term storage adjust to pH 7–8 with HEPES, and add 1 mM EDTA. Store at 4░C or –20░C. 2. 3. DNA Isolation Procedure DNA is isolated from the interphase and phenol-chloroform layer saved from the Phase separation step. DNA precipitation 1. Remove any remaining aqueous phase overlying the interphase. This is critical for the quality of the isolated DNA. Add 0.3 mL of 100% ethanol per of 1 mL TRIzol« Reagent used for the initial homogenization. Cap the tube and invert the sample several times to mix. Incubate samples for 2–3 minutes at room temperature. Centrifuge the tube at 2000 Î g for 5 minutes at 4░C to pellet the DNA. Remove the phenol-ethanol supernatant and save it in a new tube if protein isolation is desired. The supernatant can be stored at –70░C for several months. Proceed with the DNA wash step using the DNA pellet. Wash the DNA pellet with 1 mL of sodium citrate/ ethanol solution (0.1 M sodium citrate in 10% ethanol, pH 8.5) per 1 mL of TRIzol« Reagent used for the initial homogenization. Incubate for 30 minutes at room temperature. Mix occasionally by gentle inversion. Note: The DNA can be stored in sodium citrate/ethanol solution at least 2 hours. Centrifuge at 2000 Î g for 5 minutes at 4░C. Remove and discard supernatant. Repeat wash (steps 1–3), once. Note: Repeat wash twice for large DNA pellets (>200 Ág). Add 1.5–2 mL 75% ethanol per 1 mL of TRIzol« Reagent used for the initial homogenization. Note: DNA samples may be stored in 75% ethanol at 4░C for several months. Incubate for 10–20 minutes at room temperature. Mix the tube occasionally by gentle inversion. Centrifuge at 2000 Î g for 5 minutes at 4░C. Remove and discard supernatant. Air or vacuum dry the DNA pellet for 5–10 minutes. Do not allow the pellet to dry out. Do not dry the pellet by vacuum centrifuge. Proceed to the DNA resuspension step. Determining Yield of RNA and DNA Use absorbance of RNA and DNA at 260 nm and 280 nm to determine concentration. Sample RNA Procedure 1. Dilute sample in RNase-free water, and measure absorbance at 260 nm and 280 nm. 2. Use the formula A260 Î dilution Î 40 = μg RNA/mL to determine concentration. 1. Dilute sample in water or buffer (pH >7.5), and measure absorbance at 260 nm and 280 nm. 2. Use the formula A260 Î dilution Î 50 = μg DNA/mL to determine concentration. 2. 3. 4. 5. 6. DNA 7. 1. DNA wash Expected yields The table below presents typical yields of RNA (A260/280 of >1.8) and DNA (A260/280 of 1.6–1.8) from various starting materials. Starting Material Epithelial Cells New Tobacco Leaf Fibroblasts Cultured cells, mammal Skeletal muscles and brain Placenta Liver Kidney Quantity 1 Î 106 cells — 1 Î 106 cells 1 Î 106 1 mg 1 mg 1 mg 1–1.5 Ág 1–4 Ág 6–10 μg 3–4 μg RNA 8–15 Ág 73 Ág 5–7 Ág DNA — — 5–7 Ág 5–7 Ág 2–3 Ág 2–3 Ág 3–4 μg 3–4 μg 2. 3. 4. 5. Protein Isolation Procedure Proteins are isolated from the phenol-ethanol supernatant layer left over after the DNA precipitation step. Isolate the protein using either Protein precipitation OR Protein dialysis. 6. 7. 8. Protein precipitation 1. Add 1.5 mL of isopropanol to the phenol-ethanol supernatant per of 1 mL TRIzol« Reagent used for the initial homogenization. Incubate samples for 10 minutes at room temperature. Centrifuge at 12,000 Î g for 10 minutes at 4░C to pellet the protein. Remove and discard the supernatant. 9. 2. 3. 4. Proceed to the Protein wash step with the remaining protein pellet. Protein wash Prepare a wash solution consisting of 0.3 M guanidine hydrochloride in 95% ethanol. 2. Wash the protein pellet with 2 mL of the wash solution per 1 mL of TRIzol« Reagent used for the initial homogenization. 3. Incubate for 20 minutes at room temperature. Note: Protein samples may be stored in 0.3 M guanidine hydrochloride-95% ethanol for at least one month at 4░C or for at least one year at –20░C. 4. Centrifuge at 7500 Î g for 5 minutes at 4░C. Remove and discard the wash solution. 5. Repeat steps 2–4, two more times. 6. Add 2 mL of 100% ethanol to protein pellet after the third wash and vortex. 7. Incubate for 20 minutes at room temperature. 8. Centrifuge at 7500 Î g for 5 minutes at 4░C. Remove and discard ethanol wash. 9. Air dry the protein pellet for 5–10 minutes. Do not allow the pellet to dry out. 10. Proceed to the Protein resuspension step. 1. Protein resuspension, continued Poor solubility of the pellet in SDS can occur, because the solubility of specific classes of proteins differs with different solvents. If the protein pellet is insoluble in SDS, the following alternative solvents (Hummon et. al., 2007) may be required to solubilize the pellet: • • • • • 1. 1% SDS and 62.5 mM sarkosyl at pH 8.0–8.8 9.5 M urea and 2% CHAPS, pH 9.1 250mM glycerol, 10mM TEA, and 4% CHAPS 2% diethylamine 10M Urea Load the phenol-ethanol supernatant into the dialysis membrane. Note: The phenol-ethanol solution can dissolve some types of dialysis membranes (e.g., cellulose ester). Test dialysis tubing with the membrane to assess compatibility before starting. Dialyze the sample against 3 changes of 0.1% SDS at 4░C. Make the first change of solution after 16 hours, the second change 4 hours later (at 20 hours), and the final change 2 hours later (at 22 hours). Note: 0.1% SDS is required to resolubilize the proteins from the pellet; a lower concentration of SDS is insufficient. If desired, the SDS can be diluted after solubilization. Centrifuge the dialysate at 10,000 Î g for 10 minutes at 4░C. Proteins are located in the clear supernatant. Transfer supernatant to a new tube and proceed to downstream application, or store the sample at –20░C. (Optional) Solubilize the pellet by adding 100 μL of 1% SDS and 100 μL of 8 M urea. Protein dialysis 2. Protein resuspension 1. Add 1% SDS to the protein pellet (200 μL) and pipet up and down until the protein is resuspend. Note: To completely dissolve the protein pellet, you may need to incubate the sample at 50░C in a water bath or heat block. Centrifuge at 10,000 Î g for 10 minutes at 4░C to sediment any insoluble material. Transfer the supernatant containing the protein to a new tube and proceed to downstream application of choice, or store the sample at –20░C. 3. 4. 5. 2. 3. Determining Yield of Protein Measure protein concentration by Bradford assay (SDS concentration must be <0.1%). Troubleshooting Problem Low yield of RNA, Low yield of DNA, or Low yield of protein Cause • Incomplete homogenization or lysis of samples • Final RNA, DNA or protein pellet was incompletely redissolved RNA is degraded, DNA is degraded, or Protein is degraded. RNA contamination or DNA contamination • Samples were not immediately processed or frozen after collection. • Isolated RNA, DNA, or protein preparations were stored at the incorrect temperature. • Interphase/organic phase pipetted up with aqueous phase. • Incomplete removal of aqueous phase • DNA pellet insufficiently washed with 0.1 M sodium citrate in 10% ethanol Low A260/280 for RNA • Sample was homogenized in an insufficient volume of TRIzol« Reagent • Incomplete removal of organic phase Phenol was not sufficiently removed from the DNA preparation Solution • Decrease the amount of starting material. Mince tissues into smaller pieces and make sure it is completely immersed in TRIzol« Reagent for optimal lysis. • Increase the solubilization rate by pipetting the sample repeatedly, and heat the sample to 50–60░C. • Sample must be processed or frozen immediately after collection. • Store RNA samples at –60 to –70░C. Store DNA and protein samples at –20░C. • Do not attempt to draw off the entire aqueous layer after phase separation. • Remove remnants of the aqueous phase prior to DNA precipitation. • Make sure pellet is washed with 0.1 M sodium citrate in 10% ethanol. • Add the appropriate amount of TRIzol« Reagent for your sample type (see Homogenizing samples). • Do not attempt to draw off the entire aqueous layer after phase separation. Wash the DNA pellet one additional time in 0.1 M sodium citrate in 10% ethanol. Low A260/280 for DNA References: Chomczynski, P. (1993) A reagent for the single-step simultaneous isolation of RNA, DNA and proteins from cell and tissue samples. BioTechniques 15, 532-537 Chomczynski, P., and Sacchi, N. (1987) Single Step Method of RNA Isolation by Acid Guanidinium Thiocyanate-Phenol-Chloroform Extraction. Anal. Biochem. 162, 156-159 Hummon, A. B., Lim S. R., Difilippantonio, M. J., Ried, T. (2007) Isolation and solubilization of proteins after TRIzol« extraction of RNA and DNA from patient material following prolonged storage. BioTechniques 42, 467-472 Limited Use Label License No. 358: Research Use Only: The purchase of this product conveys to the purchaser the limited, non-transferable right to use the purchased amount of the product only to perform internal research for the sole benefit of the purchaser. No right to resell this product or any of its components is conveyed expressly, by implication, or by estoppel. This product is for internal research purposes only and is not for use in commercial services of any kind, including, without limitation, reporting the results of purchaser’s activities for a fee or other form of consideration. For information on obtaining additional rights, please contact [email protected] or Out Licensing, Life Technologies, 5791 Van Allen Way, Carlsbad, California 92008. ®2010 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners. TRIzol« is a registered trademark of Molecular Research Center, Inc.
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