TRI Reagent® DNA/Protein Isolation Protocol

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

• Phenol phase and interphase + 0.3 ml ethanol (per 1 ml TRI Reagent)

• 1 ml DNA Wash Solution, 2 x 30 min
• 1.5–2 ml 75% ethanol

• 8 mM NaOH, then adjust pH

• 200–500 μl phenol-ethanol supernatant (1 volume) + acetone (3 volumes)

• 1 ml Protein Wash 1, 3 x 10 min
• 1 ml Protein Wash 2, 1 x 10 min

• 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

• 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.
  

• 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 10 7 cells
• 0.1 M or 1 M HEPES (free acid), see Table 1
  

• 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.

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.

Remove any aqueous phase remaining over the interphase
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.

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

• 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
   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.

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

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.

• 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.

• 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