一种改良的植物DNA提取方法

【关键词】:DNA条形码;;改良CTAB法;;植物DNA提取;;提取方法
【摘要】:植物组织中含有大量多糖、多酚、酯类等次生代谢产物,要从中提取高质量的DNA比较困难。针对这一情况,该文提出一种改良CTAB植物DNA提取方法(mCTAB),并以10种常见植物为实验材料,与4种常用的植物DNA提取试剂盒作对比。结果表明,mCTAB法提取的DNA产率高且质量好,PCR扩增成功率也较高,而提取成本显著低于DNA提取试剂盒,可有效用于植物DNA条形码等研究的植物DNA提取。
It is very important but usually difficult to extract high quality DNA from plants for molecular work since there exist a great deal of polysaccharides, hydroxybenzenes, esters and other secondary metabolities. In this paper we pro-vide a simple modified CTAB (mCTAB) protocol for extracting plant DNA. The mCTAB method protocol includes 18 steps. (1) Weigh ca. 20 mg of dry plant tissue and ground into powder with sand using a mortar or a pestle. Remove the powder into a 2.0 mL microcentrifuge tube. (2) Add 1.0 mL pre-cooled buffer A (Table 2) to the tube, mix well and incubate the tube on ice for 15 min. Mix sample 2-3 times during incubation by inverting the tube. (3) Centrifuge the tube at 7 000 ×g for 10 min. Discard the supernatant liquid by pouring it out of the tube. (4) Repeat step 2 and 3 until the supernatant is not viscous. (5) Add 0.7 mL buffer B (Table 3), mix well and incubate at 65°C for 90–120 min. Mix the sample several times during incubation by inverting the tube. (6) Centrifuge at 10 000 ×g for 10 min, remove the supernatant to a new micro-centrifuge tube. The precipitate is reusable from step 5 if necessary. (7) Add 0.7 mL CI (chloroform: isoamyl alcohol=24:1, v/v), mix it well for 10 min by inverting tube gently. (8) Centrifuge at 10 000 ×g, for 10 min, carefully remove the super-natant to a new 1.5 mL microcentrifuge tube. (9) Repeat step 7 and 8 until no precipitate appearing between the two lay-ers of liquid after centrifuging. (10) Add 0.5 mL pre-cooled isopropanol, carefully mix well . Incubate at –20°C for 20 min. (11) Centrifuge at 10 000 ×g for 10 min, discard the supernatant, centrifuge the tube briefly to collect the remaining liquid and remove it by pipetting. (12) Add 0.1 mL RNase (100 mg·L –1 ) and incubate at 37°C for 30–60 min. (13) Add 0.1 mL ddH 2 O, 0.1 mL 5 mol·L -1 NaCl and 0.8 mL pre-cooled ethanol (95%), carefully mix well. (14) Centrifuge at 10 000 ×g for 10 min, discard the supernatant. (15) Add 0.5 mL 75% ethanol, re-suspend the pellet, centrifuge at 10 000 ×g for 2 min, discard the supernatant. (16) Repeat step 15. (17) Add 0.1 mL TE to dissolve DNA after ethanol has evaporated. (18) Estimate the concentration and the purity of the DNA solution. Store it at 4°C for immediate use, at -20°C for short time storage and -80°C for long time storage. We compared our protocol with four frequently used and commercially available kits. The result showed that our mCTAB method yielded much more DNA of high quality that is suitable for PCR amplifi-cation but with much lower cost.