CTAB PROTOCOL FOR DNA EXTRACTION

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Materials

CTAB (Cetyl Trimethyl Ammonium Bromide) buffer, Chloroform: Iso Amyl Alcohol (24:1), 7.5 M Ammonium/Sodium Acetate, Isopropanol (ice cold), 70 % Ethanol (ice cold), Molecular grade water.

Constituents of the CTAB Buffer

2% CTAB, 100 mM Tris HCl (pH=8), 20 mM EDTA (Ethylenediaminetetraacetic acid), 1.4 M NaCl, 0.2% β-mercaptoethanol – added just before use, 1% PVP (polyvinyl Pyrrolidone); make up the volume of the buffer with water.

Cell Lysis

- Homogenize 100 mg of sample in 300 μl CTAB buffer
- Transfer the homogenates into 1.5ml micro-centrifuge tubes and incubate at 60oC for 30 min.

DNA Purification

- After incubation, centrifuge the homogenates at 12000 x g for 5 min and then transfer the supernatant to a clean micro-centrifuge tube.
- Add 250 μl of Chloroform: Isoamyl Alcohol (24:1) and mix for 2 min by inverting the tube.
- Centrifuge at 14000 x g for 10 min.
- Transfer the upper aqueous phase only (contains the DNA) to a clean micro-centrifuge tube.
- Add 1 μl RNase and incubate at 37oC for 30 minutes.
- To each tube add 30 μl of 7.5 M Ammonium Acetate followed by 300 μl of ice cold Isopropanol, then invert the tubes slowly several times to mix and then leave for 2 hr or overnight at room temperature to precipitate the DNA.
- Centrifuge at 14000 x g for 15 min to pellet the DNA precipitate, and then carefully remove the supernatant.
- Wash the DNA with 300 μl of ice cold 70% ethanol, and centrifuge at 14000 x g for 1 min.  Remove the supernatant and repeat the wash step.
- Remove all supernatant and allow the DNA pellet to dry at room temperature.
- Re-suspend the DNA in 50 μl of molecular grade water.

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