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EXTRACTION OF PROTEIN_ RNA_ AND DNA USING THE TRIZOL_ PROTOCOL

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EXTRACTION OF PROTEIN_ RNA_ AND DNA USING THE TRIZOL_ PROTOCOL Powered By Docstoc
					EXTRACTION OF PROTEIN, RNA, AND DNA USING THE TRIZOL®
PROTOCOL (LAB 3/4/08)
      TRI Reagent® is a complete and ready-to-use reagent for the isolation of total RNA
or the simultaneous isolation of RNA, DNA and proteins from samples of human, animal,
plant, yeast, bacterial and viral origin. We will use it to extract these macromolecules
from bee larvae (kindly provided by Dr. Hranitz). Today we will focus on isolating and
characterizing the total RNA fraction, saving the others for later analysis.

      TRI Reagent combines phenol and guanidine thiocyanate in a mono-phase solution
to facilitate the immediate and most effective inhibition of RNase activity. A biological
sample is homogenized or lysed in TRI Reagent and the homogenate is separated into
aqueous and organic phases chloroform addition and centrifugation. RNA remains
exclusively in the aqueous phase, DNA in the interphase and proteins in the organic
phase. RNA is precipitated from the aqueous phase by addition of isopropanol, washed
with ethanol and solubilized. DNA and proteins are sequentially precipitated from the
interphase and the organic phase with ethanol and isopropanol, washed with ethanol and
solubilized in water.
NOTE: RNAse contamination is a common problem. Liberally treat the work area with
RNASEAway™. Each bench will have 1 liter of DEPC-treated water from which all of
your solutions are to be made. DEPC (diethyl pyrocarbonate) is an RNAse inhibitor.
Because TRI Reagent® is toxic, and your hands are contaminated with RNAses, use
gloves for all manipulations.

STAGES OF RNA ISOLATION OVERVIEW:
1.   HOMOGENIZATION: Bee larvae
2.   PHASE SEPARATION: homogenate + 0.2 ml chloroform.
3.   RNA PRECIPITATION: aqueous phase + 0.5 ml isopropanol.
4.   RNA WASH: 1 ml 75% ethanol.
5.   RNA SOLUBILIZATION: water.

DETAILED PROTOCOL:
        1. HOMOGENIZATION: Weigh larva. If less than 75 mg, combine two in the
        same 1.5 ml tube. Add 1 ml TRI Reagent® and thoroughly grind using a
        homogenizer, pipette tip, or Bead-Beater™ . Use 1 ml of TRI Reagent® per 50-
        150 mg tissue (i.e., use 1 ml for this experiment).

        2. PHASE SEPARATION:
               a. Incubate the homogenate for 5 minutes at room temperature to permit
        the complete dissociation of nucleoprotein complexes. Spin at top speed for 1m to
        remove debris. Transfer liquid supernatant to a new tube.
               b. Add 0.2 ml chloroform/ ml TRI Reagent®. Vortex for 15 seconds.
               c. Incubate @ rmT for 10m.
               d. Spin @ top speed @ 4ºC X 15m.
               e. Following centrifugation, the mixture separates into a lower red phenol-
        chloroform phase, an interphase and the colorless upper aqueous phase. RNA
        remains exclusively in the aqueous phase whereas DNA and proteins are in the
       interphase and organic phase. The volume of the aqueous phase is about 60% of
       the volume of TRI Reagent used for homogenization

3. RNA PRECIPITATION
        a. Transfer the aqueous phase to a fresh tube and save the interphase and organic
phase at 4ºC for subsequent isolation of DNA and proteins.
        b. Precipitation of RNA.
                1) Add 0.5 ml isopropanol/1.0 ml TRI Reagent® (i.e., 0.5 ml isopropanol).
                2) Mix.
                3) Incubate@RmT X 10 m.
                4) Spin @ top speed @ 4ºC X 8m.
NOTE: RNA precipitate forms a gelatinous white pellet on the side of the tube. It is often
invisible before centrifugation.

3. RNA WASH
        a. Remove the supernatant and add 1 ml 75% ethanol to resuspend pellet.
        b. Mix vigorously.
        c. Spin @ ½ top speed @ 4ºC X 5m.
NOTE: If the RNA pellet accumulates on the side of the tube and has a tendency to float,
re-spin the pellet at top speed X 5 m.

4. RNA SOLUBILIZATION
       a. Pour off the ethanol wash and briefly air-dry the pellet (3 - 5 m).
       b. Dissolve RNA with DEPC-treated water by passing the solution a few times
through a pipette tip and incubating for 10-15 minutes at 55-60 C.

4. RNA QUANTITATION AND VISUALIZATION
      a. Quantitate your sample spectrophotometrically by reading@A260.
      b. Visualize your total RNA on a 0.8% TAE agarose gel.
      c. Load 1 µg total. Heat to 65ºC X 10 m, and then add RNA Gel Loading Dye
              (~2X).
       d. Use the 1kb marker as your size standard.

NOTE: rinse your gel box with RNASEAway™, use DEPC–treated water to make 1X
TAE and agarose.

STORE THE REMAINDER OF YOUR REACTIONS (PROTEIN AND DNA
FRACTIONS) @ -20ºC IN YOUR SAMPLE BOX.

				
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