Efficient DNA Purification Techniques: Modified Phenol Extraction and Freeze Squeeze Methods

DNA Purification Techniques

Purifying DNA from gels is an essential step in molecular biology, enabling downstream applications such as cloning, sequencing, and genotyping. Among various methods, Modified Phenol Extraction and the Freeze Squeeze Method are highly effective for isolating DNA fragments from low melting point agarose gels. These techniques ensure high yield and purity, vital for accurate experimental results.

DNA purification techniques isolate DNA from cells or tissues by removing proteins, lipids, and other contaminants—essential for accurate genetic analysis and molecular biology research.

Modified Phenol Extraction of DNA from Low Melting Point Agarose

Materials and Reagents:

• Ethidium Bromide (or alternative visualization dye)
• Low Melting Point Agarose Gel
• Tris-saturated Phenol
• Chloroform:Octanol (24:1)
• 2× Buffer:
  • 400 mM NaCl
  • 20 mM Tris-Cl (pH 7.4)
  • 2 mM EDTA
• Butanol
• Ethanol (100%)
• 70% Ethanol with Acetate

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Step-by-Step Protocol:

1. Prepare Gel and Excise Bands:

   • Stain the gel briefly with ethidium bromide (30 minutes) or use indirect visualization.
   • Excise DNA bands into a plastic tube wrapped in foil.

2. Weigh and Transfer Gel Bands:

   • Weigh the gel slice and transfer it to a silanized Corex tube.

3. Prepare 2× Buffer:

   • Combine buffer components as listed and ensure thorough mixing.

4. Melt Agarose:

   • Add an equal volume of 2× buffer to the gel slice.
   • Heat at 68°C until the agarose dissolves completely. Keep the solution dark to protect the DNA.

5. Phenol Extraction:

   • Add Tris-saturated phenol in increments:
     • Add 1/3 volume of phenol to the melted agarose and vortex briefly.
     • Add the second 1/3, vortex again.
     • Add the final 1/3, allowing a precipitate to form.

6. Centrifuge and Separate Layers:

   • Spin at 10,000 rpm for 10 minutes at 4°C.
   • Transfer the supernatant to a new Corex tube, avoiding the pellet.

7. Chloroform Extraction:

   • Add 1 volume of chloroform:octanol (24:1) to the supernatant.
   • Vortex until the solution becomes milky white.
   • Let the mixture sit on ice for 1 minute to separate layers.
   • Remove the lower cloudy phase and discard.

8. Volume Reduction with Butanol:

   • Reduce the volume by approximately half using butanol extraction.

9. Final Cleanup:

   • Repeat chloroform-octanol extraction once more.
   • Precipitate DNA with ethanol and wash the pellet with 70% ethanol-acetate.

10. Dry and Resuspend DNA:

• Dry the pellet and resuspend in the desired volume of TE buffer.

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Freeze Squeeze Purification of DNA Restriction Fragments

Materials and Reagents:

• Phenol
• Phenol:Chloroform
• 3 M Sodium Acetate
• 100% Ethanol
• TE Buffer

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Step-by-Step Protocol:

1. Prepare Gel and Excise Bands:

   • Run DNA on an agarose gel.
   • Stain and visualize DNA bands under UV light.
   • Cut the gel slices corresponding to the bands and place them in 15 mL tubes.

2. Freeze and Mash Gel Fragments:

   • Freeze the tubes for 5–10 minutes.
   • Mash the gel slices using a flat-ended glass rod.

3. Phenol Extraction:

   • Add 100 µL of phenol per gel slice.
   • Vortex for 10 seconds.
   • Freeze at -20°C for 30 minutes (or -70°C for 5–15 minutes).
   • Centrifuge at room temperature for 15 minutes.

4. Phase Separation:

   • Remove the upper aqueous layer and transfer it to a clean tube.

5. Repeat Phenol Extraction:

   • Add an equal volume of phenol to the aqueous layer.
   • Vortex for 10 seconds and centrifuge for 5 minutes.
   • Collect the upper aqueous phase.

6. Phenol:Chloroform Extraction:

   • Extract the aqueous phase once with phenol:chloroform.

7. DNA Precipitation:

   • Add 10 µL of 3 M sodium acetate per 100 µL of supernatant.
   • Add 2 volumes of cold 100% ethanol.
   • Incubate at -20°C for at least 2 hours.

8. Pellet and Wash DNA:

   • Centrifuge for 5 minutes to pellet the DNA.
   • Wash the pellet with 70% ethanol to remove impurities.

9. Dry and Resuspend DNA:

   • Dry the DNA pellet and resuspend in 10 µL of TE buffer.

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Comparison of Techniques

Aspect	Modified Phenol Extraction	Freeze Squeeze Method
Complexity	Moderately complex	Simple and straightforward
Yield	High	Moderate
Time Required	4–6 hours	2–3 hours
Suitability	Larger DNA fragments	Small to medium DNA fragments
Cost	Moderate	Low

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Both the Modified Phenol Extraction and Freeze Squeeze Method are reliable techniques for purifying DNA fragments from agarose gels. The choice between the two depends on the size of the DNA fragments, time constraints, and experimental goals. By following these protocols, researchers can achieve high-purity DNA suitable for downstream applications like sequencing, cloning, or PCR.