CTAB 植物核酸萃取溶液
CTAB 植物核酸萃取溶液可以广泛用於植物组织的 DNA 萃取
CTAB 植物核酸萃取溶液
CTAB Extraction Buffer
CTAB 植物核酸萃取溶液可以广泛用於植物组织的 DNA 萃取,多糖类 (Polysaccharides) 以及多酚 (polyphenols) 通常是萃取植物 DNA 时最棘手的问题,CTAB 植物核酸萃取溶液利用其阳离子试剂 (cationic detergent CTAB, hexadecyltrimethylammonium bromide/ cetyltrimethylammounium bromide) 来去除多糖类和多酚并透过 Polyvinylpyrrolidone 来结合多酚,来达成降低萃取污染物的步骤。
传统的 CTAB 核酸萃取会先将样品加入 CTAB 溶液,而后进行样品均质化作用,接著离心将杂质及多糖类沉淀下来,取上清液再进行 chloroform 萃取及酒精沉淀等步骤即可完成萃取,使用 CTAB 来进行植物样品萃取通常可以获得非常纯净的产物。
CTAB 萃取植物 DNA 的建议实验步骤
- Pulverize 100 mg of plant sample using a liquid nitrogen chilled mortar and pestle. Once processed, mix 100 mg of frozen powdered sample with 500 µl of CTAB Plant Extraction Buffer.
- Place the homogenate into a 60°C bath for 30 min.
- Centrifuge the homogenate for 10 minutes at 10,000 x g.
- Transfer the supernatant into a clean tube and add 5 µl of RNase (10 mg/ml in water) to the lysate. Incubate at room temperature for 15 minutes.
- Centrifuge for 5 minutes at 10,000 x g.
- Extract the lysate with equal volume of chloroform: isoamyl alcohol (24:1). Vortex for 5 seconds then centrifuge for a minute at 10,000 x g to separate the phases.
- Transfer the upper phase to a clean tube.
- Repeat step 7 until upper layer is clear. Transfer upper phase to a new tube.
- Add 0.7 volumes of isopropanol. Mix and incubate at -20°C for 15 minutes.
- Centrifuge for 10 minutes at 10,000 x g. Decant and wash the pellet with 70% ethanol.
- Decant without disturbing the pellet. Dry the pellet briefly in the SpeedVac (3 minutes at medium heat). Do not over dry the DNA.
- Resuspend the DNA in 50 µl of TE buffer.
如果希望不使用 chloroform 的方法,可以参考不需 chloroform 的植物专用萃取试剂套组