CTAB-based DNA extraction for citrus

2×CTAB solution: 2% (w/v) CTAB, 100mMTris-HCl pH8.0, 1.4M NaCl, 20mM EDTA pH8.0 [1]* High-salt precipitation solution: 1.2M NaCl , 0.8M Sodium citrate [2]

• 10 mg/ml RNase (Nippon gene)
• Chloroform
• Isopropanol
• 70% Ethanol
• TE buffer: 10 mM Tris-HCl, 1 mM EDTA, pH 8.0 Reference

1. Allen GC, Flores-Vergara MA, Krasynanski S et al. A modified protocol for rapid DNA isolation from plant tissues using cetyltrimethylammonium bromide. Nat Protoc 2006;1:2320–5.
2. Chomczynski P, Mackey K. Modification of the TRI Reagent procedure for isolation of RNA from polysaccharide- and proteoglycan-rich sources. Biotechniques 1995;19:492–5.

Preheat 2×CTAB solution to 60°C in water bath. Add 2% (v/v) of 2-Mercaptoethanol to the 2×CTAB solution just before use.

Homogenize 100mg of fresh leaf in liquid Nitrogen. Add 800µL of 2×CTAB solution and completely suspend homogenized leaf. Transfer the suspended solution to 2 ml tube.

Add4µL of 10mg/mL RNase, mix by inversion, and incubate at 37°C for 0h 15m 0s (In order to prevent RNase contamination in laboratory, RNase treatment is conducted before denaturing proteins by chloroform).

Incubate at 56°C for 0h 30m 0s inverting the tube once every 0h 10m 0s.

Add 300µL of Chloroform and mix gently with tube rotator for 0h 15m 0s.

13000rpm,25°C

Transfer supernatant carefully to new 2 ml tube.

Repeat step 6 and 7

Transfer 600µL of supernatant to new 1.5 ml tube, add 300µL of High-salt precipitation solution, and mix by inversion.

Add 300µL of Isopropanol and mix by inversion.

15000rpm,25°C

Discard and remove supernatant (DNA pellet is often transparent).

Add 1000µL of 70% ethanol and mix by inversion 10 times to wash salts.

15000rpm,25°C

Completely remove supernatant and dry up DNA pellet in air.

Dissolve pallet in 100µL of TE buffer.