植物学报2013，Vol.48Issue(1)：72-78,7.DOI:10.3724/SP.J.1259.2013.00072
一种改良的植物DNA提取方法
A Modified CTAB Protocol for Plant DNA Extraction
摘要
Abstract
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 provide 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℃ for 90-120 min. Mix the sample several times during incubation by inverting the tube. (6) Centrifuge at 10 000 xg 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 Cl (chloroform: isoamyl alcohol=24:1, v/v), mix it well for 10 min by inverting tube gently. (8) Centrifuge at 10 000 xg, for 10 min, carefully remove the supernatant to a new 1.5 mL microcentrifuge tube. (9) Repeat step 7 and 8 until no precipitate appearing between the two layers of liquid after centrifuging. (10) Add 0.5 mL pre-cooled isopropanol, carefully mix well . Incubate at -20℃ for 20 min.(11) Centrifuge at 10 000 xg 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℃ for 30-60 min. (13) Add 0.1 mL ddH2O, 0.1 mL 5 mol·L-1NaCI and 0.8 mL pre-cooled ethanol (95%), carefully mix well. (14) Centrifuge at 10 000 xg for 10 min, discard the supernatant. (15) Add 0.5 mL 75% ethanol, re-suspend the pellet, centrifuge at 10 000 xg 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℃ for immediate use, at -20℃ for short time storage and -80℃ 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 amplification but with much lower cost.关键词
DNA条形码/改良CTAB法/植物DNA提取/提取方法Key words
DNA barcoding/ mCTAB/ plant DNA extraction/ protocol引用本文复制引用
李金璐,王硕,于婧,王玲,周世良..一种改良的植物DNA提取方法[J].植物学报,2013,48(1):72-78,7.基金项目
科技部科技基础性工作专项(No.2011FY120200-6)、高技术研究发展计划(No.2012AA021602)、国家科技支撑计划(No.2012BAC01B05-6)和东北林业大学研究生科技创新基金 （No.2011FY120200-6）
