Intact Chloroplast Isolation by Sucrose Gradient Centrifugation and cpDNA Extraction

Intact chloroplast organelles were isolated using the sucrose gradient method (Takamatsu et al. 2018). Some 20 g of fresh leaves from each plant were frozen in liquid nitrogen and macerated. The material was resuspended in 200 ml of isolation buffer (50 mM Tris–HCl [pH 8.0], 0.35 M sucrose, 7 mM ethylenediaminetetraacetic acid, 5 mM 2-mercaptoethanol, and 0.1% bovine serum albumin) and incubated for 10 min in the dark. The suspension was filtered through two layers of gauze and then two layers of Miracloth (Merck), and the filtrate centrifuged at 1,000 × g for 10 min. Finally, the pellet was washed in 50 ml of isolation buffer and centrifuged at 1,000 × g for 10 min.

The pellet was resuspended in 5 ml of isolation buffer and the suspension slowly laid out in a 20/45% sucrose density batch gradient in 50 mM Tris–HCl (pH 8.0), 0.3 M sorbitol, and ethylenediaminetetraacetic acid 7 mM. The gradient was centrifuged at 2,000 × g for 30 min. The green band formed at the interface containing the intact chloroplasts was collected, diluted with three volumes of isolation buffer, and centrifuged at 3,000 × g for 10 min to obtain the purified chloroplasts in the pellet.

The pellet was resuspended in 2% CTAB buffer to promote lysis. The suspension was then incubated at 65 °C for 1 h with stirring. The supernatant was extracted 2× with an equal volume of chloroform:isoamyl alcohol (24:1) and centrifuged at 10,000 × g for 20 min. An equal volume of isopropanol was added to the upper layer and incubated at 20 °C for 1 h. Finally, the aqueous phase was centrifuged at 10,000 × g for 20 min, and the cpDNA pellet washed with ethanol (70%), dried, and resuspended in 40 μl of Tris–ethylenediaminetetraacetic acid buffer.