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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

  1. 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.
  2. Place the homogenate into a 60°C bath for 30 min.
  3. Centrifuge the homogenate for 10 minutes at 10,000 x g.
  4. 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.
  5. Centrifuge for 5 minutes at 10,000 x g.
  6. 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.
  7. Transfer the upper phase to a clean tube.
  8. Repeat step 7 until upper layer is clear. Transfer upper phase to a new tube.
  9. Add 0.7 volumes of isopropanol. Mix and incubate at -20°C for 15 minutes.
  10. Centrifuge for 10 minutes at 10,000 x g. Decant and wash the pellet with 70% ethanol.
  11. Decant without disturbing the pellet. Dry the pellet briefly in the SpeedVac (3 minutes at medium heat). Do not over dry the DNA.
  12. 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

 


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