A.Isolation and Purification

A newly devised procedure utilizing liquid nitrogen［4］is used for isolation of chloroplasts and purification of ct-DNA.This method minimizes the damage of nuclei，and therefore greatly reduces contamination with nuclear DNA.The yield of ct-DNA from this procedure is also substantially higher.

Tobacco seedlings of about I inch are transplanted into 5-inch soil pots in the greenhouse.The plants are allowed to grow for 4 to 8 weeks（depending on the seasons after transplantation）.The leaves are harvested after being placed in the dark for 2 days in order to deplete them of starch.

此文发表于1982年“Plant Molecular Biology Newsletter”Ⅲ：95-100.(https://www.daowen.com)

After deveining，100 to 500 g of freshly harvested leaves are homogenized with liquid nitrogen in a metal waring blender.After the liquid is evaporated and the powder is warmed at room temperature to-5℃，three weight volumes of isolation buffer（A）（0.33 M Sorbitol，5 mM MgCI，50 mM Tris-HCI（pH 8.0），0.1%BSA and 0.mM mercaptoethanol）is added to the powder with rapid mixing.The suspension is filtered through miracloth and centrifuged at 1，500 g for 15 min.The crude chloroplast pellets are then gently resuspended in the same buffer and purified through a discontinuous silica sol gradient［5］containing 0.24 M sucrose throughout.A clear band of chloroplasts，which appear to be mostly intact，as observed under microscope，is located in the middle layer，whereas the nuclei and mitochondria are distributed in the bottom and top layers respectively.The chloroplast band is collected，mixed with an equal volume of buffer A，and pelleted at 12，000 g for 30 min.The pellet is resuspended sequentially in 3.6 ml of buffer B（50 mM Tris，20 mM EDTA，pH 8.0），0.9 ml of 2%Na-Sarkosyl（in 50 mM Tris，10 mM EDTA，pH 8.0），and 1.5 ml of 4 M CsCI（in 50 mM Tris，10 M EDTA，pH=8.0）in order to lyse chloroplasts.

The chloroplast lysate is pelleted at 1，200 g for 30 min，after which the supernatant is removed and supplemented by 2.6 g of CsCI and 0.1 ml of ethidium bromide（10 mg/ml）.Another extraction of the pellet with lysing buffers listed above always provides a higher yield of ct-DNA.7—8 ml of the supernatant is layered on to 3 ml of CsCI（ρ=1.74）in nitrocellulose tubes，then centrifuged at 40，000 rpm for 18 hours at 20℃.After centrifugation the single fluorescent ct-DNA band is visualized by ultraviolet illumination.The ct-DNA band is collected and pooled，and the ethidium bromide is removed by shaking with 0.01 M EDTA-saturated isoamyl alcohol.The DNA is dialysed at 4℃against three 500 ml changes of buffer C（50 mM Tris，20 mM NaAc，2 mM EDTA，18 mM NaCI）over a 24 hours period.The ct-DNA concentration is usually between 10 and 100 μg/ml after dialysis.The ct-DNA is precipitated with 2 volumes of ethanol and resuspended in buffer D（10 mM Tris，I mM EDTA，pH 8.0）to ct-DNA concentration of 1μgμd.This ct-DNA is ready for restriction pattern analysis and molecular cloning.