Nucleic Acids Extraction

7.2.1. Nucleic Acids Extraction#

Author:

Hailing Fang

Created:

20240709

Updated:

20240709

Genomic DNA Extraction from Animal Cells#

Genomic DNA Extraction with Phenol-Chloroform#

Protocol ID:

p0001

Objective

Theory

Materials and Reagents

Reagents

Phenol; Chloroform; Isoamyl alcohol; TE buffer (Tris-EDTA); Proteinase K; RNase A; Centrifuge; Ethanol or isopropanol;

Procedure

  1. Cell Lysis:

    Resuspend cells in a lysis buffer containing SDS and Proteinase K to digest proteins and lyse the cells.

  2. Phenol-Chloroform Extraction:

    Add an equal volume of phenol:chloroform

  3. alcohol (25:24:1).

    Vortex and centrifuge to separate phases.

  4. Aqueous Phase Collection:

    Carefully collect the upper aqueous phase (containing DNA) without disturbing the interface.

  5. Chloroform Extraction:

    Add an equal volume of chloroform, vortex, and centrifuge again to remove any remaining phenol.

  6. DNA Precipitation:

    Add cold ethanol or isopropanol to the aqueous phase to precipitate the DNA. Incubate at -20°C for 30 minutes or overnight.

  7. DNA Collection:

    Centrifuge to pellet the DNA, wash with 70% ethanol, and air dry the pellet.

  8. DNA Resuspension:

    Resuspend the DNA pellet in TE buffer or water.

Total RNA Extraction from Cells#

Contents