Procedure

Genomic DNA isolation from Arabidopsis thaliana seeds:

Step 1:

Mature dry Arabidopsis seeds or Arabidopsis seeds that have been imbibed for a germination assay and are at stages prior to the young seedling stage (i.e., 48–72 h after seed imbibition under normal conditions).

Step 2:

Keep the Reagents for DNA extraction ready
a. CTAB B solution: 1.4 M NaCl, 0.1 M Tris–HCl (pH 8.0), 2% (w/v) CTAB. Store at room temperature.
b. CTAB C solution: 50 mM Tris–HCl (pH 8.0), 10 mM EDTA (pH 8.0), 1% (w/v) CTAB. Store at room temperature.
c. CTAB D solution: 50 mM Tris–HCl (pH 8.0), 10 mM EDTA (pH 8.0), 1 M CsCl, 1% (w/v) CTAB. Store at room temperature.

Step 3:

Freeze seed material (maximum 100 mg of dry seeds) with liquid N2 and grind with a mortar and pestle. Transfer material to a 2 ml microcentrifuge tubes and add 750 microlitre of CTAB B solution.

Step 4:

Incubate for 10 min at 65°C with occasional vortexing. Add 500 ul of a 1:1 mixture of CTAB B and CTAB C. Incubate for 10 min at 65°C with occasional vortexing.

Step 5.

Centrifuge at 18,000×g for 15 min at room temperature. Transfer the supernatant to a clean tube and add 500 ul of chloroform. Mix contents gently, but thoroughly, and centrifuge at 18,000×g for 5 min at room temperature.

Step 6:

Repeat steps of centrifugation and chloroform extraction. Transfer the upper phase to a fresh tube and add one volume of CTAB C. Mix thoroughly.

Step 7:

Incubate for 30 min at room temperature. Remove the supernatant and resuspend pellet in 500 mL of CTAB D supplemented with 1 mg of RNase A. Incubate for 30 min at 37°C.

Step 8:

Add two volumes of 100% ethanol. Allow the DNA to precipitate for 20 min at −20°C. Then centrifuge at 18,000×g for 5 min at room temperature. Remove supernatant and wash pellet in 75% ethanol. Then centrifuge at 18,000×g for 5 min at 4°C.

Step 9:

Remove supernatant and resuspend pellet in 50 ul TE buffer. Estimate DNA concentration by measuring absorbance at 260 nm (OD260) using a spectrophotometer. At this point, DNA concentrations are in the range of 200 ng/mL to 1 mg/mL