Plant DNA extraction – CTAB Prep

About 1 g of Arabidopsis leaves is ground (in a microfuge tube) to a fine powder using liquid nitrogen. When dry ice has all sublimed away, hot (65oC) 2x CTAB buffer (1:1v/v, about 200μl) is added and mixed well. The homogenate is incubated in a water bath (65oC) for 10 min. About 200μl (1:1 v/v) of Chloroform: Isoamyl alcohol (24:1 v/v) is added and mixed well. The sample is centrifuge at 4000 rpm for 10 min. Supernatant is transferred into a new tube and about 20 μl of 10 % CTAB-NaCl buffer (65oC) is added and mixed well. Chloroform: Isoamyl step is repeated. About 800 μl (2:1 v/v) CATB precipitation buffer is added and mixed gently for 5 to 10 min. Tube is kept in 0oC bath for 1 hour. Then the sample is centrifuge at 13000 rpm for 5min. Supernatant is discarded. About 500 μl of high salt TE buffer is added and kept in 65oC water bath till pellet dissolves. About 1ml of cold ethanol (100 %) is added and mixed well. If DNA is not precipitated, tube is kept in -20oC for 30 min. Sample is centrifuged at 13000 rpm for 5 min and supernatant is discarded. Pellet is washed with 75 % ethanol (1 ml) and air dried. Pellet is rehydrated in 0.1xTE buffer.

Solutions

(1)  2X CATAB buffer :

  1. 2% CATB (w/v)
  2. 100 mM Tris (pH 8.0)
  3. 20 mM EDTA (pH 8.0)
  4. 1.4 M Nacl
  5. 1% PVP (polyvinylpyrrolidone)

(2)  10% CTAB solution:

  1. 10% CTAB
  2. 0.7 M NaCl

(3)  CTAB precipitation buffer

  1. 1% CTAB
  2. 50 mM Tris (pH 8.0)
  3. 10 mM EDTA (pH 8.0)

(4)  High-salt TE buffer:

  1. 10mM Tris (pH 8.0)
  2. 1 mM EDTA (pH 8.0)
  3. 1 M NaCl

(5)  0.1XTE buffer

  1. 1.0 mM Tris (pH 8.0)
  2. 0.1 mM EDTA (pH 8.0)