CTAB法小量制备植物基因组DNA

1、CTAB法小量制备植物基因组DNA【原理】 CTAB分离DNA的方法最初用于细菌，后来经修改用于从植物中获取DNA。CTAB（即十六烷基三乙基溴化铵）是一种阳离子去污剂。CTAB可与核酸形成复合物，该复合物仅溶于高盐溶液。此外，CTAB可十分有效地促进DNA和RNA与多糖的分离并去除多糖。可通过提高NaCl的浓度和沉淀核酸去除CTAB。残留的CTAB可用76%80%乙醇洗涤核酸沉淀去除；CTAB较易溶于乙醇因而可通过洗涤溶液去除。某些植物组织包含高浓度的酚类物质。除去这些化合物至关重要，因为酚类化合物可氧化形成醌，后者可与核酸交联。聚乙烯吡咯烷酮（PVP）和-巯基乙醇（BME）裂解混合液中的常

2、见组分，是有效的还原剂，可将因酚类化合物氧化形成的醌降至最低。PVP在某种程度上相当于一种巨大的多糖，本质上它可占据物理空间从而增加其它分子的有效浓度并使得这些分子的沉淀更为容易。任何基因组DNA制备技术的目的是分离足够纯的、高分子量的DNA。有两种因素会影响所分离DNA的尺寸：剪切力和核酸酶活性。因此裂解应当温和处理从而将剪切力降至最小。植物细胞富含核酸酶。大多数核酸酶需要Mg2+作为辅因子。为了降低核酸酶活性，组织应当快速冷冻并在包含有去污剂和高浓度EDTA的提取缓冲液中解冻。 【材料】植物组织，新鲜液氮2× CTAB提取缓冲液氯仿:异戊醇【24:1（v/v）】异丙醇洗涤缓冲液9

3、5%乙醇TE 缓冲液（pH 8.0）10 mg/mL 无DNase的RNase A 金属匙研钵和研杵37°C和65°C水浴微量离心机移液器涡旋仪微型真空泵记号笔灭菌的1.5 mL一次性微量离心管灭菌吸头手套【步骤】1. 称取100 mg叶片组织，并在液氮预冷的研钵中研磨为细粉末。然后将粉末转移至1.5 mL微量离心管中。 2. 添加700 L新鲜的2× CTAB提取缓冲液并用涡旋仪混匀。3. 样品置于65°C水浴锅中30分钟，并每隔15分钟摇动离心管。将样品冷却至室温。4. 每管添加700 L氯仿:异戊醇（24:1），然后颠倒混匀数次。5. 室温14,0

4、00 rpm离心5分钟。将上层水相转移至新的、已标记的1.5 mL微量离心管中。 æ 注意：低于15°C将致使CTAB-核酸复合物沉淀。6. 重复步骤4和5。(可以根据实际过程增减此步骤)7. 向每管中添加等体积、4°C的异丙醇（约400500 L）。颠倒混匀，然后将管置于4°C、30分钟或者20°C、15分钟。8. 14,000 rpm离心10分钟。小心地弃去上清液避免丢失DNA沉淀。倒置离心管并在空气中干燥。9. 用1 mL洗涤缓冲液清洗沉淀（约3分钟），14,000 rpm离心10分钟。小心弃去上清液。10. 用1 mL 95%乙醇洗涤沉

5、淀、于14,000 rpm离心10分钟，然后小心地弃去乙醇。倒置离心管在空气中干燥或者用真空泵抽吸15分钟。 11. 每个样品用2050 L TE溶解DNA，并添加12 L无DNase的RNase A（10 mg/mL）。37°C孵育30分钟。 12. 确定浓度后，贮存于4°C（或长期贮存于20°C）。Small-scale Preparation of Plant Genomic DNA Using CTABPrinciple The CTAB method of DNA isolation was initially used in bacteria and

6、later modified to obtain DNA from plants. CTAB (cetyltrimethylammonium bromide) is one of cationic detergents. CTAB forms a complex with nucleic acids, and the complex is only soluble in high salt. Moreover, CTAB quite efficiently promotes the separation of both DNA and RNA from polysaccharides, and

7、 remove polysaccharides. CTAB is removed by raising the NaCl concentration and precipitating the nucleic acids. The residual CTAB is removed by washing the nucleic acid pellet with 76-80% ethanol; CTAB is more soluble in ethanol and is discarded with the wash solution.Tissues from some plants contai

8、n high concentrations of phenolics. Elimination of these compounds is essential because phenolic compounds form quinones upon oxidation which crosslink nucleic acids. PVP and BME, common lysis buffer components, are effective reducing reagents, which will minimize quinone formation due to the oxidat

9、ion of phenolic compounds. PVP is a rather large polysaccharide that essentially takes up physical space, thereby increasing the effective concentration of other molecules and making it easier to precipitate them. The aim of any genomic DNA preparation technique is to isolate high-molecular-weight D

10、NA of sufficient purity. Two factors affect the size of the DNA isolated: shear forces and nuclease activity. So lysates should be treated gently to minimize shear forces. Plant cells are rich in nucleases. Most nucleases require Mg2+ as a cofactor. To reduce nuclease activity, the tissue should be

11、frozen quickly and thawed only in the presence of an extraction buffer that contains detergent and a high concentration of EDTA.Materials Plant tissue, freshLiquid nitrogen2× CTAB extraction buffer24:1 (v/v) chloroform: isoamyl alcoholIsopropanolWash buffer95% EtOHTE buffer, pH 8.010 mg/mL DNas

12、e-free RNase A Metallic spoonMortar and pestle37°C and 65°C water bathMicrocentrifugePipettorsVortexerMini-vacuum pumpMarker penSterile 1.5 mL disposable microcentrifuge tubesSterile tipsGloves Procedure1. Weigh 100 mg of leaf tissue and grind into a pre-chilled mortar in liquid nitrogen t

13、o obtain a fine powder. Transfer powder to a 1.5 mL microcentrifuge tube. 2. Add 700 L of fresh 2× CTAB extraction buffer and vortex. 3. Place samples in a 65°C water bath for 45 min, shaking tubes every 15 min. Cool samples at RT.4. Add 700 L of chloroform: isoamyl alcohol (24:1) to each

14、tube. Then invert tubes several times.5. Spin at 14,000 rpm for 5 min in a microcentrifuge at RT. Remove aqueous top layer and transfer it to a new, labeled 1.5 mL microcentrifuge tube. æ Note: Below 15°C the CTAB/nucleic acid complex may precipitate.6. Repeat Steps 4 and 5.7. Add an equal

15、 volume of 4°C isopropanol (400-500 L) to each tube. Invert tubes several times and let tubes sit at 4°C for 30 min or at 20°C for 15 min.8. Spin at 14,000 rpm for 10 min. Pour off the supernatant carefully to avoid losing the DNA pellet. Invert tubes and air-dry.9. Wash the DNA pellet with 1 mL of wash buffer (for 3 min) and spin at 14,000 rpm for 10 min. Carefully pour off the supernatant. 10. Wash the pellet in 1 mL of 95% EtOH, spin for 10 min at 14,000 rpm, and carefully pour off the EtOH. Invert tubes and air-dry overnight or use a vacuum pump for 1