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                    Products > RNA Purification > TRAzol (R1021/R1022)

                    TRAzol (R1021/R1022)


                    Reagents required, but not supplied:
                    ? Chloroform
                    ? Isopropyl alcohol
                    ? 75% Ethanol (in DEPC-treated water)
                    ? DEPC-treated water

                    The  TRAzol RNA Purification Kit provides a simple, reliable, and rapid  method for isolating high–quality total RNA from a wide variety of  samples, including animal and plant cells and tissue, bacteria, and  yeast. The kit utilizes the strong lysis capability of TRAzol Reagent.  TRAzol Reagent maintains the integrity of the RNA, while disrupting  cells and dissolving cell components. Addition of chloroform followed by  centrifugation, separates the solution into an aqueous phase and an  organic phase. RNA remains exclusively in the aqueous phase. After  transfer of the aqueous phase, the RNA is recovered by precipitation  with isopropyl alcohol. After removal of the aqueous phase, the DNA and  proteins in the sample can be recovered by sequential precipitation.  Precipitation with ethanol yields DNA from the interphase, and an  additional precipitation with isopropyl alcohol yields proteins from the  organic phase. Copurification of the DNA may be useful for normalizing  RNA yields from sample to sample. Total RNA isolated by TRAzol Reagent  is free of protein and DNA contamination. It can be used for Northern  blot analysis, dot blot hybridization, poly (A)+ selection, in vitro translation, RNase protection assay, and molecular cloning.

                    The most classic formula
                    The most widely used
                    The most stable yield


                    Store at 2-8°C, protect from light for up to 12 months.

                    Precautions for Preventing RNase Contamination
                    RNases  can be introduced accidentally into the RNA preparation at any point in  the isolation procedure through improper technique. Because RNase  activity is difficult to inhibit, it is essential to prevent its  introduction. The following guidelines should be observed when working  with RNA.
                    Always wear disposable gloves. Skin often contains bacteria  and molds that can contaminate an RNA preparation and be a source of  RNases. Practice good microbiological technique to prevent microbial  contamination.
                    Use sterile, disposable plasticware and automatic  pipettes reserved for RNA work to prevent cross-contamination with  RNases from shared equipment. For example, a laboratory that is using  RNA probes will likely be using RNase A or T1 to reduce background on  filters, and any nondisposable items (such as automatic pipettes) can be  rich sources of RNases.

                    Recommended volume of TRAzol on different starting materials

                    10 cm2 adherent cells

                    1 ml

                    107 suspension cells

                    1-2 ml

                    100 μl white cells

                    2 ml

                    50-100 mg ordinary tissue

                    1 ml

                    50-100 mg special tissue(live, spleen, bone or cartilage)

                    2 ml

                    15-100 mg plant tissue

                    1 ml

                    Lysate Preparation with TRAzol Reagent
                    Use TRAzol Reagent to prepare lysates from various sample types as described below.

                    tissue  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  TRAzol Reagent per 50–100 mg tissue using a tissue homogenizer or  rotor-stator.

                    Adherent Cells
                    Lyse cells directly in a culture  dish by adding 1 ml of TRAzol Reagent to the dish and passing the cell  lysate several times through a pipet tip. The amount of TRAzol Reagent  required is based on the culture dish area (1 ml per 10 cm2) and not on the number of cells present.

                    Suspension Cells
                    Harvest cells and pellet cells by centrifugation. Use 1 ml of the TRAzol Reagent per 5–10 × 106 animal, plant, or yeast cells, or per 1 × 107  bacterial cells. Lyse cells by repetitive pipetting up and down. Do not  wash cells before addition of TRAzol Reagent to avoid any mRNA  degradation. Disruption of some yeast and bacterial cells may require a  homogenizer.

                    Phase Separation
                    Following cell or tissue lysis (previous protocol), perform the following steps to isolate the RNA.
                    1.  Incubate the lysate with TRAzol Reagent (previous protocol) at room  temperature for 5 minutes to allow complete dissociation of  nucleoprotein complexes.
                    2. Add 0.2 ml chloroform per 1 ml TRAzol Reagent used. Shake the tube vigorously by hand for 15 seconds.
                    Note:  Vortexing may increase DNA contamination of your RNA sample. Avoid  vortexing if your downstream application is sensitive to the presence of  DNA.
                    3. Incubate at room temperature for 2–3 minutes.
                    4. Centrifuge the sample at 12,000× g for 5-10 min at 4°C.
                    Note:  After centrifugation, the mixture separates into a lower, red  phenol–chloroform phase, an interphase, and a colorless upper aqueous  phase which contains the RNA.
                    5. Transfer of the colorless, upper phase containing the RNA to a fresh RNase–free tube.
                    6.  Add an equal volume of Isopropyl alcohol. Invert the tube to disperse  any visible precipitate that may form after adding ethanol.
                    7. Centrifuge at 12,000× g for 15 seconds at room temperature.

                    RNA Wash
                    Discard  the supernatant, add l ml 75% Ethanol (in DEPC-treated water), do not  stir the precipitate, gentlely invert the tube several times to wash the  tube, centrifuge at 12,000 x g for 2 min at 4 °C, discard the ethanol,  repeat the step again.

                    Redissolving the RNA
                    Dry  the tube containing the RNA precipitate 2–5 minutes, do not centrifuge  or heat to dry, or the RNA will be difficult to dissolve. Add  appropriate volume of Rnase-free water to dissolve the precipitate,  pipetting if it is necessary to completely dissolve the RNA. Store at  -80℃.


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