DNA purification is a crucial step in molecular biology to isolate high-quality DNA for downstream applications like PCR, cloning, and sequencing. This article outlines a detailed DNA purification protocol alongside the role of each chemical involved, ensuring an understanding of both the process and the science behind it.


DNA Purification Protocol

Step 1: RNA Degradation

  • Add 5 µl of RNase (100 mg/ml) to the sample.
  • Incubate at 37°C for 10 minutes to digest the RNA.

Step 2: Deproteinization

  • In a fume hood, mix 5 ml phenol with 5 ml chloroform/isoamyl alcohol (24:1).
  • Shake vigorously. This step denatures proteins and aids in separating nucleic acids from proteins and other cellular components.

Step 3: Centrifugation

  • Centrifuge at 4000 rpm for 8 minutes.
  • Carefully decant the upper aqueous phase into a fresh labeled tube — this phase contains the DNA.

Step 4: DNA Precipitation

  • Add 2 volumes of ethanol and gently invert. White strands of high-molecular-weight DNA may appear.
  • Centrifuge at 4000 rpm for 10 minutes, then discard the ethanol.

Step 5: Washing the DNA Pellet

  • The pellet may appear white (ideal) or clear (less stable).
  • Carefully remove residual ethanol with a pipette.

Step 6: Drying and Resuspension

  • Air-dry the pellet with the tube on its side.
  • Resuspend the pellet in 100-200 µl of TE buffer (pH 8).

Step 7: Final Cleanup

  • Perform a final chloroform/isoamyl alcohol (24:1) extraction to remove remaining phenol.
  • Adjust volume to 300-400 µl with TE buffer and add an equal volume of chloroform/IAA.
  • Invert and centrifuge for 5 minutes in a microcentrifuge.

Step 8: DNA Re-precipitation

  • Add 2 volumes of 100% ethanol, invert gently, and centrifuge again for 8 minutes.
  • Discard ethanol, add 500 µl of 70% ethanol, and centrifuge at full speed for 5 minutes.
  • Discard ethanol and air-dry the final pellet.
  • Resuspend in 20-50 µl TE buffer for storage.


Role of Chemicals

Liquid Nitrogen:

Freezes samples, making cells brittle for easy lysis.
Prevents heat-induced DNA denaturation during homogenization.

Tris Buffer:

Maintains pH between 6-8, stabilizing DNA.

EDTA:

Chelates divalent cations like Mg²⁺, inhibiting DNase activity to protect DNA.

NaCl:

Prevents DNA aggregation and provides Na⁺ ions to neutralize the negative phosphate backbone, enhancing solubility.

CTAB (Cetyltrimethylammonium Bromide):

Disrupts cell walls in plants and other tough tissues.

PVP (Polyvinylpyrrolidone):

Removes polyphenols that can interfere with DNA integrity (especially in plant samples).

Beta-mercaptoethanol:

Reduces disulfide bonds in proteins, aiding denaturation.

Phenol-Chloroform-Isoamyl Alcohol (24:1):

Phenol denatures proteins, which then precipitate.

Chloroform dissolves lipids and polysaccharides.

Isoamyl alcohol stabilizes the interface and reduces foaming during mixing.

Isopropanol/Absolute Ethanol:

Precipitates DNA by reducing its solubility.

70% Ethanol:
Washes excess salts without disturbing the DNA pellet.

RNase:
Specifically digests RNA, ensuring only DNA remains in the sample.


pH and Phenol’s Role in DNA/RNA Separation

  • Phenol pH > 7.0 (pH 8): DNA collects in the upper aqueous phase.
  • Phenol pH 4.5: RNA stays in the aqueous phase, while DNA and proteins collect at the interface.


Conclusion

This protocol ensures high-purity DNA isolation by carefully removing proteins, RNA, and contaminants. Each chemical has a defined role, from lysing cells to stabilizing the final DNA pellet. Understanding these roles allows for troubleshooting and optimization, ensuring reliable results for downstream molecular biology applications.

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