TRIzol® Reagent
Catalog Numbers Quantity Store at 2°C to 25°C 15596-026 100 mL
15596-018 200 mL
Description
TRIzol® Reagent is a ready-to-u 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 RNa
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 ur to perform quential 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 parate into a clear upper aqueous layer (containing RNA), an interpha, 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 interpha/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 u in downstream applications.
•Isolated RNA can be ud in RT-PCR, Northern Blot analysis, Dot Blot hybridization, poly(A)+ lection, in vitro translation, RNa protection assay, and molecular cloning.
•Isolated DNA can be ud in PCR, Restriction Enzyme digestion, and Southern Blots.
•Isolated protein can be ud 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 glass. Contact your Environmental Heath and Safety (EH&S) department for proper work and disposal guidelines. Avoid direct contact with TRIzol® Reagent, becau contact to skin, eyes, or respiratory tract may cau chemical burns to the expod area. If contact to skin or eyes occurs, immediately wash the expod area with copious amounts of water for 15 minutes and ek medical attention if necessary. If you inhale vapors, move to fresh air and ek medical attention if necessary. For more information, refer to the TRIzol® Reagent SDS (Safety Data Sheet), available from our web site at /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 U
For rearch u only. Not intended for human or animal diagnostic or therapeutic us.
Materials Needed
The following additional materials are needed, but not supplied for the isolation of RNA, DNA or proteins.
Part no. 15596026.PPS MAN0001271Rev. Date: 13 Dec 2012
For support, visit /support or email To reorder, visit
Preparing Samples Homogenizing samples 1.
at room temperature according to the table below. The
sample volume should not exceed 10% of the volume of
TRIzol® Reagent ud for homogenization. Be sure to u
the indicated amount of TRIzol® Reagent, becau an
insufficient volume can result in DNA contamination of
isolated RNA.
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 subquent DNA isolation on your sample.
3.Proceed to Pha paration, or store the homogenized
sample. Homogenized samples can be stored at room
temperature for veral hours, or at –60 to –70°C for at least
one month.
Incubate the homogenized sample (e Homogenizing
samples) for 5 minutes at room temperature to permit
complete dissociation of the nucleoprotein complex.
2.Add 0.2 mL of chloroform per 1 mL of TRIzol® Reagent
ud for homogenization. Cap the tube curely.
3.Shake tube vigorously by hand for 15 conds.
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4.Incubate for 2–3 minutes at room temperature.
5.Centrifuge the sample at 12,000 × g for 15 minutes at 4°C.
Note: The mixture parates into a lower red phenol-
chloroform pha, an interpha, and a colorless upper
aqueous pha. RNA remains exclusively in the aqueous pha.
The upper aqueous pha is ~50% of the total volume.
6.Remove the aqueous pha of the sample by angling the
tube at 45° and pipetting the solution out. Avoid drawing
any of the interpha or organic layer into the pipette when
removing the aqueous pha.
7.Place the aqueous pha into a new tube and proceed to
the RNA Isolation Procedure.
8.Save the interpha and organic phenol-chloroform pha
if isolation of DNA or protein is desired. See DNA
Isolation Procedure and Protein Isolation Procedure for
details. The organic pha can be stored at 4°C overnight.
RNA Isolation Procedure
Always u the appropriate precautions to avoid RNa
contamination when preparing and handling RNA.
RNA precipitation
1.(Optional) When precipitating RNA from small sample
quantities (<106 cells or <10 mg tissue), a dd 5–10 µg of
RNa-free glycogen as a carrier to the aqueous pha.
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.
2.Add 0.5 mL of 100% isopropanol to the aqueous pha, per
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1 mL of TRIzol® Reagent ud for homogenization.
3.Incubate at room temperature for 10 minutes.
4.Centrifuge at 12,000 × 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.
5.Proceed to RNA wash.
RNA wash
1.Remove the supernatant from the tube, leaving only the
RNA pellet.
2.Wash the pellet, with 1 mL of 75% ethanol per 1 mL of
TRIzol® Reagent ud 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.
3.Vortex the sample briefly, then centrifuge the tube at
7500 × g for 5 minutes at 4°C. Discard the wash.
4.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, becau
the pellet can lo solubility. Partially dissolved RNA
samples have an A260/280 ratio <1.6.
5.Proceed to RNA resuspension.
RNA resuspension
1.Resuspend the RNA pellet in RNa-free water or
0.5% SDS solution (20–50 μL) by passing the solution up
and down veral times through a pipette tip.
Note: Do not dissolve the RNA in 0.5% SDS if it is to be
ud in subquent enzymatic reactions.
2.Incubate in a water bath or heat block t at 55–60°C for
10–15 minutes.
3.Proceed to downstream application, or store at –70°C. DNA Isolation Procedure
DNA is isolated from the interpha and phenol-chloroform layer saved from the Pha paration step.
DNA precipitation
1.Remove any remaining aqueous pha overlying the
interpha. This is critical for the quality of the isolated
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DNA.
2.Add 0.3 mL of 100% ethanol per of 1 mL TRIzol® Reagent
ud for the initial homogenization.
3.Cap the tube and invert the sample veral times to mix.
4.Incubate samples for 2–3 minutes at room temperature.
5.Centrifuge the tube at 2000 × g for 5 minutes at 4°C to
pellet the DNA.
6.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 veral months.
7.Proceed with the DNA wash step using the DNA pellet. DNA wash
1.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 ud for the initial
homogenization.
2.Incubate for 30 minutes at room temperature. Mix
lake baikaloccasionally by gentle inversion.
Note: The DNA can be stored in sodium citrate/ethanol
solution at least 2 hours.
3.Centrifuge at 2000 × g for 5 minutes at 4°C. Remove and
discard supernatant.
4.Repeat wash (steps 1–3), once.
Note: Repeat wash twice for large DNA pellets (>200 µg).
5.Add 1.5–2 mL 75% ethanol per 1 mL of TRIzol® Reagent
ud for the initial homogenization.
Note: DNA samples may be stored in 75% ethanol at 4°C
for veral months.
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6.Incubate for 10–20 minutes at room temperature. Mix the
tube occasionally by gentle inversion.
7.Centrifuge at 2000 × g for 5 minutes at 4°C. Remove and
discard supernatant.
8.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.
9.Proceed to the DNA resuspension step.DNA resuspension
Resuspend the DNA in 8mM NaOH at a concentration of
0.2–0.3 µg/µL.
职业化心态1.Add 0.3–0.6 mL of 8mM NaOH per 50–70 mg of tissue,
or per 1 × 107 cells.
英文书写格式Note: Resuspending the DNA is a mild ba is highly
recommended becau isolated DNA does not
resuspend well in water or Tris buffer.
2.Remove any insoluble material by centrifuging the
sample at 12,000 × 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
日语网站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.
Determining Yield of RNA and DNA
U absorbance of RNA and DNA at 260 nm and 280 nm to determine concentration.
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Expected yields
The table below prents typical yields of RNA (A260/280 of
>1.8) and DNA (A260/280 of 1.6–1.8) from various starting materials.
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. Protein precipitation
1.Add 1.5 mL of isopropanol to the phenol-ethanol
supernatant per of 1 mL TRIzol® Reagent ud for the
initial homogenization.
2.Incubate samples for 10 minutes at room temperature.
3.Centrifuge at 12,000 × g for 10 minutes at 4°C to pellet the
protein. Remove and discard the supernatant.
4.Proceed to the Protein wash step with the remaining
protein pellet.
Protein wash
1.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 ud 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 × 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 × 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.
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.
2.Centrifuge at 10,000 × g for 10 minutes at 4°C to diment any
英文翻译中文在线翻译insoluble material.
3.Transfer the supernatant containing the protein to a new tube
and proceed to downstream application of choice, or store the sample at –20°C. Protein resuspension, continued
Poor solubility of the pellet in SDS can occur, becau the solubility
of specific class 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% 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
Protein dialysis
1.Load the phenol-ethanol supernatant into the dialysis
membrane.
Note: The phenol-ethanol solution can dissolve some types of
dialysis membranes (e.g., cellulo ester). Test dialysis tubing
with the membrane to asss compatibility before starting.
2.Dialyze the sample against 3 changes of 0.1% SDS at 4°C. Make
the first change of solution after 16 hours, the cond 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.
3.Centrifuge the dialysate at 10,000 × g for 10 minutes at 4°C.
Proteins are located in the clear supernatant.
4.Transfer supernatant to a new tube and proceed to downstream
application, or store the sample at –20°C.
5.(Optional) Solubilize the pellet by adding 100 μL of 1% SDS and
100 μL of 8 M urea.
Determining Yield of Protein
Measure protein concentration by Bradford assay (SDS
concentration must be <0.1%).
Troubleshooting
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 U Label Licen No. 358: Rearch U Only: The purcha of this product conveys to the purchar the limited, non-transferable right to u the purchad amount of the product only to perform internal rearch for the sole benefit of the purchar. No right to rell this product or any of its components is conveyed expressly, by implication, or by estoppel. This product is for internal rearch purpos only and is not for u in commercial rvices of any kind, including, without limitation, reporting the results of purchar’s activities for a fee or other form of consideration. For in
formation on obtaining additional rights, plea contact or Out Licensing, Life Technologies, 5791 Van Allen Way, Carlsbad, California 92008.
©2010 Life Technologies Corporation. All rights rerved. The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners.
TRIzol® is a registered trademark of Molecular Rearch Center, Inc.
