protocol

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Introduction

Procedure Overview

The following protocols from Molecular Research Center, Inc., (MRC) describe sequential isolation of DNA and protein from the interphase and organic/phenol phase of the lysate from the TRI Reagent® * RNA Isolation Protocol.

The RNA Isolation Protocol is provided with TRI Reagent (Cat #9738) and is available at: http://www.ambion.com

The DNA isolated by this protocol is suitable for PCR, restriction enzyme digestion, and Southern blotting. The recovered protein is suitable for analysis by Western blotting.


DNA Isolation Protocol

a. DNA Precipitation
  • Phenol phase and interphase + 0.3 ml ethanol (per 1 ml TRI Reagent)
b. DNA Wash
  • 1 ml DNA Wash Solution, 2 x 30 min
  • 1.5–2 ml 75% ethanol
c. DNA Solubilization
  • 8 mM NaOH, then adjust pH


Protein Isolation Protocol

a. Protein Precipitation
  • 200–500 μl phenol-ethanol supernatant (1 volume) + acetone (3 volumes)
b. Protein Wash
  • 1 ml Protein Wash 1, 3 x 10 min
  • 1 ml Protein Wash 2, 1 x 10 min
c. Protein Solubilization
  • 1% SDS, 10M Urea or other suitable solvent

Protocol - TRI Reagent DNA Isolation

DNA is precipitated from the phenol phase and interphase of samples that have been homogenized (or lysed) in 1 ml of TRI Reagent (step 5 in the RNA Isolation Protocol). After a series of washes to remove residual phenol, the DNA pellet is solubilized in a mild alkaline solution, and the pH is adjusted. This technique performs well with samples containing >10 μg of DNA.

Before You Start

Required Materials
  • 100% ethanol, ACS grade or better
  • Nuclease-free water
  • Trisodium citrate
  • NaOH
  • HEPES (free acid)
  • Appropriately sized nuclease-free centrifuge tubes with secure closures, compatible with phenol/chloroform, and capable of withstanding centrifugal forces of 12,000 x g.

Reagent Preparation

  • DNA Wash Solution: 0.1 M trisodium citrate in 10% ethanol (no pH adjustment required), 2–3 ml per 1 ml of TRI Reagent used in the initial homogenization:
  • 75% ethanol, 1.5–2 ml per 1 ml TRI Reagent used in the initial homogenization
  • 8 mM NaOH,300–600 μl per 50–70 mg tissue or 107 cells
  • 0.1 M or 1 M HEPES (free acid), see Table 1

Protocol Notes

  • Unless stated otherwise, conduct the procedure at room temperature.
  • The molecular weight of the recovered DNA depends on the shearing forces applied during homogenization. If recovery of high molecular weight DNA is desired, use a loosely fitting homogenizer in the initial homogenization step of the RNA Isolation Protocol. Avoid using a Polytron homogenizer.
  • If the DNA is isolated only for quantitative purposes: a) samples can be more vigorously homogenized, including the use of a Polytron; b) the phenol phase and interphase can be stored at 4°C for a few days or at –70°C for a few months; c) the DNA can be solubilized using 40 mM NaOH instead of an 8 mM solution, and by vortexing the DNA pellet instead of pipetting.

Special Handling Precautions

The starting material for this procedure contains TRI Reagent, which contains a poison (phenol) and an irritant (guanidine thiocyanate). Contact with TRI Reagent will cause burns and can be fatal. Use gloves and other personal protection when working with TRI Reagent.

DNA Precipitation

This procedure begins with the material remaining after step 5 of the TRI Reagent RNA Isolation Protocol, i.e., the lower, red, organic/phenol phase, and the interphase. The phenol phase and interphase can be stored at 4°C overnight before proceeding with the DNA isolation procedure.

  1. Remove any aqueous phase remaining over the interphase

    2. Remove any remaining aqueous phase overlying the interphase

    NOTE Careful removal of any residual aqueous phase is critical for the quality of the isolated DNA.

    If your sample contains <10 μg DNA, see the alternate protocol

  2. Add 300 μl of 100% ethanol and mix by inversion

    • Add 300 μl of 100% ethanol per 1 ml of TRI Reagent used for the initial homogenization.
    • Mix samples by inversion.

  3. Incubate at room temp for 2–3 min, centrifuge at 2,000 x g for 5 min at 4°C, and remove the supernatant

    • Incubate the samples at room temperature for 2–3 min.
    • Centrifuge at 2,000 x g for 5 min at 4°C to sediment the DNA.
    • Remove the supernatant and store it at 4°C for subsequent protein isolation (see TRI Reagent Protein Isolation Protocol).

DNA Wash

  1. Wash the pellet twice with 1 ml DNA Wash Solution for 30 min at room temp

    2. Incubate for the recommended times for efficient removal of phenol from the DNA pellet.
    • Add 1 ml of DNA Wash Solution per 1 ml of TRI Reagent used for the initial homogenization.
    • Incubate the DNA pellet in DNA Wash Solution for 30 min at room temperature with periodic mixing
    • Centrifuge at 2,000 x g for 5 min at 4–25°C.
    • Carefully remove the supernatant.
    • Repeat steps a–d to wash the DNA pellet a second time
    • (Optional) For large pellets containing >200 μg DNA or large amounts of a non-DNA material, repeat steps a–d a third time.


  2. Wash the pellet with 1.5–2 ml of 75% ethanol for 10–20 min at room temp

    3. This ethanol wash removes pinkish color from the DNA pellet.
    • Add 1.5–2 ml of 75% ethanol per 1 ml TRI Reagent used for the initial homogenization.
    • Incubate for 10–20 min at room temperature with periodic mixing.
    • Centrifuge at 2,000 x g for 5 min at 4–25°C.
    • Centrifuge at 2,000 x g for 5 min at 4–25°C.
      STOPPING POINT - Samples suspended in 75% ethanol can be stored at 4°C for a long period of time (months).
    • Remove the ethanol wash. Remove all residual ethanol by centrifuging again briefly and removing the ethanol that is collected.

DNA Solubilization

  1. Briefly air dry the DNA pellet

    2. Briefly air dry the DNA pellet by keeping the tube open for 3–5 min at room temperature.

  2. Dissolve the DNA pellet in 8 mM NaOH

    The DNA pellet is fully solubilized in a mild alkaline solution.

    • Add an appropriate amount of 8 mM NaOH to yield a DNA concentration of 0.2–0.3 μg/μl. Typically, add 300–600 μl of 8 mM NaOH to DNA isolated from 50–70 mg of tissue or 10 7 cells.
    • Pipette up and down slowly to dissolve the DNA pellet. Samples solubilized in 8 mM NaOH can be stored overnight at 4°C.


  3. Centrifuge at 12,000 x g for 10 min and transfer the supernatant to a new tube

    • Centrifuge at 12,000 x g for 10 min to pellet any insoluble material (fragments of membranes, etc.) remaining at this stage.
    • Transfer the DNA-containing supernatant to a new tube. High viscosity of the supernatant indicates the presence of high molecular weight DNA.


  4. Adjust the pH of the DNA solution

    • Adjust the DNA solution to the desired pH using 0.1 M or 1 M HEPES (free acid). See Table 1.
    • Add EDTA to a concentration of 1 mM.

      Table 1. Adjustment of pH in DNA samples

      Use the following amounts of 0.1 M or 1 M HEPES (free acid) per 1 ml 8 mM NaOH

      Final pH 0.1 M HEPES Final pH 0.1 M HEPES Final pH 1 M HEPES
      8.486 μl7.8 117 μl7.223 μl
      8.293 μl7.5159 μl7.0 32 μl
      8.0101 μl----

      Alternatively, dialyze samples against 1 mM EDTA, pH 7–pH 8.

DNA Isolation Troubleshooting


Low yield
  • Sample homogenization or lysis was incomplete
  • Solubilization of the final DNA pellet was incomplete
A260/A280
ratio <1.70
  • Phenol was not sufficiently removed from the DNA preparation
  • Acidic water was used for the spectrophotometric measurement.
DNA degradation
  • Tissues were not immediately processed or frozen after removing from animal
  • Samples were homogenized with a Polytron or other high speed homogenizer
RNA contamination
  • Aqueous phase was carried over with the interphase and organic phase
  • DNA pellet was not sufficiently washed with DNA Wash Solution.

Assess DNA Yield and Quality

Determine the DNA concentration and purity by diluting an aliquot of the preparation in water or buffer with a pH>7.5 and reading the absorbance in a traditional spectrophotometer at 260 nm and at 280 nm.

To determine the DNA concentration in μg/ml, multiply the A 260 by the dilution factor and the extinction coefficient
(1 A 260 = 50 μg double-stranded DNA/ml).

A260 X dilution factor X 50 = μg DNA/ml

DNA yield and size can vary considerably between samples. Yield is dependent on factors such as sample type, health of the organism, and thoroughness of sample disruption. Sample handling has a strong impact on the size of the recovered DNA. The DNA yield and size expectations listed below assume ideal conditions for factors that the user can control.
 
Tissue (mammalian) DNA yield per mg tissue Size distribution 60–100 kb     0–20 kbCultured cells DNA yield per 106 cells Size distribution 60–100 kb     0–20 kb
Liver, kidney3–4 μg~70%           ~30% Human, rat, mouse
5–7 μg  >80%          <20%
Skeletal muscle, brain, placenta2–3 μg~70%           ~30%




To evaluate DNA purity, calculate the sample’s A 260/A 280 ratio. Typically, the isolated DNA is free of RNA and protein and has an A 260/A 280 ratio >1.7. Alternatively, dialyze samples against 1 mM EDTA, pH 7–pH 8.

Alternate DNA Isolation Protocols

Small samples:   <10 μg DNA

To assure full recovery of DNA from small samples (<10 μg DNA), use an acrylamide coprecipitant, e.g., Ambion’s Linear Acrylamide Cat #9520.

Follow the standard protocol with the following modifications:
  • In step II.A.1 above, add 2–8 μl of acrylamide coprecipitant to the interphase-phenol phase after removal of remaining aqueous phase. Proceed with step II.A.2.
  • In step II.B.1 above, wash the DNA/carrier pellet twice with 75% ethanol instead of DNA Wash Solution.
  • For each wash, incubate for 10 min at room temperature with intermittent mixing. Proceed with  DNA Solubilization

 Back extraction protocol

This alternative procedure replaces steps II.A–II.B of the DNA Isolation Protocol.

  1. Prepare a back extraction buffer: 4 M guanidine thiocyanate, 50 mM sodium citrate and 1 M Tris (free base).

  2. Remove any remaining aqueous phase overlying the interphase.

  3. Add back extraction buffer to the interphase-organic phase mixture. Use 500 μl of back extraction buffer per 1 ml of TRI Reagent used for the initial homogenization.

  4. Vigorously mix the sample by inversion for 15 sec and incubate for 10 min at room temperature.

  5. Centrifuge at 12,000 x g for 15 min at 4°C to separate the phases.

  6. Transfer the upper, aqueous phase containing DNA to a clean tube and save the interphase and organic phase at 4°C for subsequent protein isolation.

  7. (Optional) If the expected DNA yield is less than 20 μg, add 2–8 μl of acrylamide carrier to the aqueous phase and mix.

  8. Add 400 μl of isopropanol per 1.0 ml of TRI Reagent used for the initial homogenization to precipitate DNA from the aqueous phase. Mix the sample by inversion and incubate for 5 min at room temperature.

  9. Sediment the DNA by centrifugation at 12,000 x g for 5 min at 4–25°C and remove the supernatant.

  10. Wash the DNA pellet with 1 ml of 75% ethanol. Proceed with DNA Solubilization

TRI Reagent Protein Isolation Protocol

Protein is precipitated from the phenol-ethanol supernatant obtained after sedimentation of the DNA pellet. After a series of washes, the protein-containing pellet is solubilized in a suitable detergent-containing solvent.

TRI Reagent protein extracts prepared from rat tissues yield 50–110 μg protein/mg tissue

Before You Start

Required Materials
  • Acetone or isopropanol, ACS grade or higher quality
  • Guanidine hydrochloride
  • 100% ethanol, ACS grade or or higher quality
  • Glycerol
  • Sodium dodecyl sulfate (SDS), urea or another suitable detergent for protein solubilization
  • (Optional) Tributylphosphine (e.g., Sigma T 7567) or another reducing agent for protein solubilization

Reagent Preparation

Protein Wash 1: 300 mM guanidine hydrochloride in 95% ethanol + 2.5% glycerol (V:V), 3 ml per sample
Protein Wash 2:  ethanol containing 2.5% glycerol (V:V), 1 ml per sample

Protocol Notes

The procedure is carried out at room temperature unless stated otherwise.

Special Handling Precautions

The starting material for this procedure contains TRI Reagent, which contains a poison (phenol) and an irritant (guanidine thiocyanate). Contact with TRI Reagent will cause burns and can be fatal. Use gloves and other personal protection when working with TRI Reagent.

Protein Precipitation

  1. Transfer 200–500 μl of the supernatant from step II.A.3c to a new microcentrifuge tube

    2. Transfer 200–500 μl (1 volume) of the phenol-ethanol supernatant from step II.A.3c  to a microcentrifuge tube.

    Limit the volume of the supernatant to 200–500 μl per tube to achieve smaller, more manageable protein pellets and improved protein yield.


  2. Add 3 volumes of acetone, mix by inversion, and incubate 10 min at room temp This step precipitates protein in the sample.

    • Add 3 volumes of acetone. For example, add 750 μl of acetone to 250 μl of phenol-ethanol supernatant. Isopropanol may be used in place of acetone, however, total protein yield may be reduced by 5–10%.
    • Mix by inversion for 10–15 sec to obtain a homogeneous solution.
    • Incubate samples for 10 min at room temperature.

  3. Centrifuge at 12,000 x g for 10 min at 4°C, and discard the supernatant

    • Centrifuge at 12,000 x g for 10 min at 4°C to sediment the protein precipitate.
    • Remove and discard the supernatant.

Protein Wash

  1. Wash the protein pellet with a total of 1 ml Protein Wash 1 for 10 min at room temp

    • Add 500 μl Protein Wash 1 to the protein pellet.
    • Disperse the pellet using a pipet tip, syringe needle or a small conical Teflon pestle (Fisher K749515–0000) attached to a mechanical stirrer (~30 sec @ 800–1000 RPM).
    • After dispersing the pellet, add another 500 μl of Protein Wash 1 to the sample.
    • Incubate for 10 min at room temperature.

  2. Centrifuge the sample at 8,000 x g for 5 min, anddiscard the supernatant

    • Sediment the protein by centrifugation at 8,000 x g for 5 min.
    • Remove and discard the supernatant.

  3. Wash two more times with 1 ml Protein Wash 1

    • These washes remove residual phenol.
    • Add 1 ml Protein Wash 1 to the pellet and disperse the pellet by vortexing.
    • Incubate for 10 min at room tempterature.
    • Sediment the protein by centrifugation at 8,000 x g for 5 min, and remove and discard the supernatant.
    • Repeat steps a–c to wash a second time with 1 ml Protein Wash 1.

    STOPPING POINT -  In general, protein pellets suspended in Protein Wash 1 or in Protein Wash 2 can be stored for at least one month at 4°C or one year at –20°C. Individual proteins may display different sensitivity to long-term storage; establish optimal storage conditions for sensitive and labile proteins.

  4. Wash with 1 ml Protein Wash 2 for 10 min

    • Disperse the pellet in 1 ml Protein Wash 2.
    • Incubate for 10 min.
    • Centrifuge the sample at 8,000 x g for 5 min at 4°C.
    • Remove and discard the supernatant, invert the tube and dry the pellet for 7–10 min at room temperature.

Protein Solubilization

  1. Resuspend the pellet in 200 μl of solvent per 10–20 mg starting tissue sample


    • Add 200 μl of solvent per 10–20 mg of tissue sample to the protein pellet. Use a solvent such as 1% SDS, 10 M urea, or another suitable detergent-based solvent. The solubility and stability of specific proteins can be influenced by different detergent solutions. Determine which works best for your experimental needs by solubilizing small samples in different solvents. Addition of a reducing agent such as tributylphosphine (2.5% of solution volume) will improve protein yield in most preparations.
    • Gently disperse and solubilize the pellet for 15–20 min by “flicking” the tube or pipetting.

    STOPPING POINT
    Samples may be stored at –20˚C at this point.

  2. Prior to use, heat the protein sample for 3 min at 100°C, centrifuge at 10,000 x g for 5 min, and transfer the supernatant to a clean tube

    • If the sample has been stored at –20°C, thaw at 25°C for 10–15 min before proceeding. Solubilized protein may form insoluble aggregates during storage.
    • Prior to use in Western analysis, heat the protein sample for 3 min at 100°C.
    • Centrifuge the sample at 10,000 x g for 5 min at room temperature to sediment any insoluble material.
    • Transfer the supernatant to a clean tube.
    • Use the sample immediately for Western blotting.

Protocol - Alternate Protein Isolation

  1. Dialyze the phenol-ethanol supernatant in a suitable, regenerated cellulose dialysis tubing against three changes of 0.1% SDS at 4°C.

  2. Centrifuge the dialysate at 10,000 x g for 10 min at 4°C and transfer the clear supernatant to a fresh tube.

  3. Use the clear supernatant for Western blotting.

Protein Isolation Troubleshooting


Low yield
  • Incomplete homogenization or lysis of samples
  • Incomplete solubilization of the final protein pellet.
Protein degradation
  • Tissues were not immediately processed or frozen after removing from animal.
Band deformation in PAGE
  • Insufficient wash of the protein pellet.

References

  1. Ausubel F M, Brent R, Kingston R E, Moore D D, Seidman J G, Smith J A and Struhl K (1990) Appendix 1, in Current Protocols in Molecular Biology, vol 2, p. A.1.5, John Wiley and Sons, Inc., New York, NY.

  2. Banerjee, S, Smallwood A, Chambers AE and Nicolaides K. (2003) Quantitative Recovery of Immunoreactive Proteins from Clinical Samples Following RNA and DNA Isolation. BioTechniques, 35, 450–456.

  3. Chomczynski P (1993) A reagent for the single-step simultaneous isolation of RNA, DNA and proteins rom cell and tissue samples. BioTechniques,15, 532–537.

  4. Chomczynski P and Mackey K (1995) Modification of the TRI Reagent® DNA/Protein Isolation procedure for isolation of RNA from polysaccharide - and proteoglycan - rich sources. Biotechniques,19, 942–945.

  5. Wilfinger W, Mackey K and Chomczynski P (1997) Effect of pH and ionic strength on the spectrophotometric assessment of nucleic acid purity. BioTechniques, 22, 474–481.

  6. Wu, LC (1997) Isolation and Long-Term Storage of Proteins from Tissues and Cells Using TRIzol Reagent. FOCUS, 17, 98–100.
LT043

For Research Use Only. Not for use in diagnostic procedures.