摘要
探索优化研磨条斑紫菜叶状体提取DNA的条件,建立一套简单、快速、高效的高通量研磨破碎方法。钢珠研磨80s时的破碎效果与液氮研磨破碎效果相近,因此选用80s作为研磨时间。利用2种样品材料（干燥和新鲜）、4种研磨介质（钢珠Φ2.3mm、陶瓷珠Φ1mm、石英砂0.270～0.707mm和玻璃珠Φ0.5mm）、3个研磨介质体积梯度（0.1、0.2和0.3mL）设计研磨实验。结果发现,钢珠的破碎效果最好,玻璃珠的破碎效果最差;石英砂和玻璃珠组研磨干燥材料所得DNA完整性最好,4种研磨介质研磨新鲜材料所得DNA出现了降解,钢珠和陶瓷珠研磨干燥材料所得DNA降解最严重;研磨介质的体积对样品的破碎程度、DNA得率和DNA质量没有显著性影响;4种研磨介质研磨干燥和新鲜材料所提DNA的纯度均较高。80s的研磨实验中显示,钢珠组能有效的破碎干燥和新鲜材料,获得DNA的纯度和得率都比较高,但是DNA出现了降解。在此基础上设置了0.2mL钢珠研磨干燥和新鲜材料5～40s的时间梯度。实验结果显示,钢珠在5～40s时均能有效的破碎干燥、新鲜2种样品材料。干燥材料研磨5～40s时,DNA有明显的主条带,DNA得率随着研磨时间的延长有所增加,DNA质量较好,且能用于限制性内切酶酶切实验;新鲜材料研磨5～40s时,DNA有明显的主条带,DNA得率没有显著性差异,DNA质量较好,也能用于限制性内切酶酶切实验。所以本文认为破碎干燥和新鲜2种样品材料制备高质量条斑紫菜DNA的最佳条件为：研磨介质为0.2mL钢珠、研磨速度为6 800r/min、研磨时间为5s。
The grinding protocol of DNA extraction from the blades of Pyropia yezoensis was optimized to build a simple, rapid, efficient and high-throughput method. Firstly, the effect of grinding for 80 s with steel bead is similar to that of grinding with liquid nitrogen, we chose the method of grinding for 80 s. In the next step, two kinds of samples （fresh and dry）, four types of beads （steel bead with 2.3 mm, porce- lain bead with 1 mm, silica bead with 0. 270-0. 707 mm and glass bead with 0. 5 mm） and three different beads volume （0. 1, 0. 2 and 0. 3 mL） were chosen to design the experiment. The efficiency of grinding with steel bead is the best, but that with glass bead is the worst the integrality of DNA from the dried blades grinded by silica bead and glass bead is the best, the DNA extracted using fresh samples grinded by those four grinding beads showed degradation, the DNA extracted from dried samples grinded by steel bead and porcelain bead degraded worsely～ the volume of grinded media had no significant affect with the size of fraction, yield and quality of the DNA; the purity of DNA extracted from dried and fresh samples grinded by those four grinding media are high. It is efficient to grind dried and fresh samples with steel bead during 80 s, the purity and yield of DNA are high too, but it showed the degradation of the DNA. From above, we used 0. 2 mL steel bead to grind dried and fresh samples with the time from 5～40 s. Ac- cording the results, we can see that it is efficient to grind both dried and fresh samples with steel bead du- ring 5～40 s. When we grinded the fresh samples for 5～40 s, it showed main site of the DNA, and the yield of DNA is higher with the time added, the quality of DNA is better and the DNA can be used for re- striction digestiom it also showed main site of the DNA when we grinded the fresh samples for 5～40 s, the yield of DNA had no significant difference, the quality of DNA was better and the DNA could also be used for restriction digestion. We hold that the optimum homogenized conditions for dried and fresh samples were.. 0. 2 mL steel bead, 6 800 r/min, and grinding for 5 s.
出处
《中国海洋大学学报（自然科学版）》
CAS
CSCD
北大核心
2014年第11期45-51,共7页
Periodical of Ocean University of China
基金
国家自然科学基金项目(31372517)
国家高技术研究发展计划项目(2012AA10A411
2012AA10A406
2012AA10A401)资助
关键词
生物样品均质器
条斑紫菜
DNA制备
bead beating homogenizer
Pyropia yezoensis
DNA extraction protocol
作者简介
田知海（1985一），男，硕士生，主要从事细胞遗传学研究。E-mail：137top@163．com
通讯作者：E-mail：yxmao@ouc．edu．cn
