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Regulatory requirements vary by product. Please see product-specific literature to determine product use.

Can I use TRIzol Reagent to isolate RNA from tissue that was stored in RNAlater Reagent? Product FAQ

Answer

Yes, tissue stored in RNAlater Reagent can be used in the TRIzol Reagent. Remove the tissue from RNAlater Reagent, and immediately submerge in TRIzol solution.

Answer Id:: E6345

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What chloroform do you recommend I use for RNA extraction with TRIzol Reagent? Are there any substitutes I can use? Product FAQ

Answer

We recommend using straight chloroform. No isoamyl alcohol is needed (though using chloroform:isoamyl alcohol 49:1 works without problems). You can also use chloroform with 50 ppm amylene. Alternatively, BCP (1-bromo-2 chloropropane) can be used in the place of chloroform.

Answer Id:: E7834

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Do you have recommendations for homogenizing my sample for RNA isolation with TRIzol Reagent? Product FAQ

Answer

You can homogenize your sample using a glass Teflon or power homogenzier (Polytron or Tekmar's Tissumizer) in a 1.5 microcentrifuge tube. Cultured cells do not have to be homogenized. Sonication will work to lyse cells in TRIzol reagent, but should only be performed if you do not plan on isolating DNA from your sample. Cells grown in monolayers can be lysed directly in the culture dish.

Answer Id:: E6346

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Can I use acetone instead of isopropanol to isolate protein using TRIzol reagent? Product FAQ

Answer

Yes, proteins can be precipitated by the addition of isopropanol or acetone. Optimal protein yield can be achieved with an acetone:phenol-ethanol ratio between 3:1 and 6:1.

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What is the difference between the TRIzol Plus RNA Purification Kit and TRIzol Reagent? Product FAQ

Answer

The TRIzol Plus RNA Purification Kit combines the lysis capability of TRIzol Reagent with the convenient RNA extraction technology of the silica spin columns included in the PureLink RNA Mini Kit.

Answer Id:: E7835

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At what points can I stop in the protocol for TRIzol Reagent? Product FAQ

Answer

There are a couple of possible stopping points in the RNA extraction protocol as shown below:

•After homogenization (before addition of chloroform), samples can be stored at 4 degrees C overnight or at –70 degrees C for at least 1 year.
•Homogenized samples can sit at room temperature for several hours before chloroform is added.
•Homogenized samples can be thawed and refrozen prior to use (necessary when researcher intends to do experiment, but then cannot continue).
• After RNA precipitation, during RNA wash, the RNA can be stored in 75% ethanol for at least 1 year at –20 degreesC, or at least 1 week at 4 degrees C.

For DNA extraction, the phenol phase and interphase can be stored at 4 degrees C overnight before DNA precipitation. Some customers have tried storing at 4 degrees C for a week and –20 degrees C for a year and still got good recovery.

Answer Id:: E6347

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Can I use TRIzol Reagent to isolate single-stranded DNA? Product FAQ

Answer

Yes, single-stranded DNA will separate with the DNA phase.

Answer Id:: E7676

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Can I skip the phase separation step in your TRIzol-based RNA prep kits? Product FAQ

Answer

All our TRIzol-based RNA prep kits, including the Phasemaker Tubes (Cat. No. A33248) include a chloroform-based phase separation step that cannot be skipped.

Answer Id:: E18632

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What carrier do you recommend for isolation of RNA with TRIzol Reagent? Product FAQ

Answer

Glycogen can be included with your sample to improve yield, and remains with the RNA (glycogen is water soluble). Polyacrylamide can also be used as a carrier to precipitate small amounts of RNA.

Answer Id:: E6348

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What is the composition of your TRIzol Reagent? Product FAQ

Answer

TRIzol Reagent is a ready-to-use mixture of phenol, guanidine isothiocyanate, red dye, and other proprietary components.

Answer Id:: E6342

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Why is the A260/280 ratio of my sample, extracted using the TRIzol Reagent, low? Product FAQ

Answer

Most likely the sample was homogenized in an insufficient volume of TRIzol Reagent. Make sure to add the appropriate amount of TRIzol Reagent for your sample type.
Alternatively, the organic phase could be incompletely removed. Do not attempt to draw off the entire aqueous layer after phase separation.

Answer Id:: E17608

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I don't see an RNA pellet after the TRIzol isolation procedure? Do you have any tips? Product FAQ

Answer

You can add glycogen to your sample, which can help improve yield and remains with the RNA (glycogen is water soluble). Polyacrylamide can also be used as a carrier to precipitate small amounts of RNA. Alternatively, you can also use salmon sperm DNA. It should be added during the precipitation of the aqueous phase.

Answer Id:: E7866

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What is the difference between TRIzol and TRIzol LS Reagent? Product FAQ

Answer

TRIzol LS Reagent is a complete, ready-to use reagent for easy and simultaneous isolation of total RNA, DNA, and proteins from liquid samples. The reagent, a mono-phasic solution of phenol and guanidinium thiocyanate, is an improvement to the single-step RNA isolation method developed by Chomzcynski and Sacchi. TRIzol LS Reagent is similar to the original TRIzol Reagent in composition and results of use. TRIzol LS Reagent is designed for use with liquid samples such as blood and virus preparations in which large volumes of aqueous samples need to be processed, while TRIzol Reagent is designed for cell cultures or tissues. It is formulated to accommodate processing of more liquid sample per unit of reagent compared to the original formula.

The only difference between TRIzol Reagent and TRIzol LS Reagent is the concentration of components. TRIzol  LS Reagent is slightly more concentrated. The formula allows lower quantities of reagent to be used relative to a liquid sample. (TRIzol = 10:1 required, TRIzol LS = 3:1 required). If you buy TRIzol LS Reagent, but want to use it like TRIzol Reagent (on solid samples), there will probably be a decrease in yield vs. using regular TRIzol Reagent. TRIzol LS Reagent should NOT be used undilted with solid samples. To dilute: take 750 µL TRIzol LS Reagent + (50 to 100 mg tissue + water to make 250 µL).

Answer Id:: E4065

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I inadvertently added isopropanol instead of chloroform. What should I do? Product FAQ

Answer

If isopropanol is inadvertently added at this step instead of chloroform, add more isopropanol to precipitate everything, then resuspend the pellet in TRIzol Reagent and use the protocol as specified. RNA yields will be compromised, but it may be possible to obtain a product in RT-PCR. A detailed protocol follows:

(1) Add more isopropanol so that the total volume of isopropanol equals the volume of TRIzol Reagent used. Spin at 7500 x g for 10 min at 4 degrees C.
(2) Pour off supernatant; allow relatively compacted pellet to air dry (doesn't have to be completely dry, just reduce the volume of ispropanol).
(3) Estimate the size of the pellet in microliters; add at least 15–20 volumes of TRIzol Reagent (e.g., for a 100 µL pellet, add at least 1.5 mL TRIzol Reagent).
(4) Break the pellet up well (you may have to use a hand-held homogenizer). Store the solution for 10–15 min. at room temperature; every 5 min or so, shake it by hand to make certain it is well dispersed.
(5) Proceed with the TRIzol Reagent protocol as written (i.e., add chloroform). Results will not be optimal, but it may be possible to get a product in RT-PCR.

Answer Id:: E6349

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Can proteins that are purified with TRIzol Reagent be immunoprecipitated? Product FAQ

Answer

Yes, as long as the antibody used binds to the target protein in its denatured form.

Answer Id:: E2966

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