大量–高纯–高浓度DNA的制备
Production of DNAs in Large-Quantity, High-Purification, and High-Concentration
DOI: 10.12677/AMB.2022.112008, PDF,  被引量    国家自然科学基金支持
作者: 邵玉强, 金家铖, 李雪刚：河南农业大学生命科学学院，河南 郑州；刘亮伟*：河南农业大学生命科学学院，河南 郑州；农业部农业酶工程重点实验室，河南 郑州
关键词: 大量；高纯；高浓度；DNA制备；Large-Quantity； High-Purification； High-Concentration； DNA Production
摘要: 氯化铯(CsCl)梯度离心能够大量制备DNA，柱回收试剂盒能够快速制备DNA，结合二者优势开发大量–高纯–高浓度DNA的制备方法。本研究以长度5.9 kb的PCR产物为材料，通过CsCl-EB溶液75,000 rpm离心6 h、注射器收集目标DNA、柱回收去除EB、CsCl回收得到DNA。2000 μL PCR产物CsCl离心后、1个柱回收洗脱四次得到22.1 μg DNA，A260/A230纯度指标高达1.99。3000 μL优化PCR产物CsCl离心后、6个柱回收，洗脱一次得到DNA浓度高达391 ng/μL，洗脱四次得到DNA量高达61.5 μg。与之相比，胶回收2000 μL PCR产物仅得到9.6 μg DNA、A260/A230指标仅0.29，远低于纯净DNA指标2，而柱回收2000 μL PCR产物有较多非特异性条带。CsCl离心–柱回收方法能够制备大量–高纯–高浓度DNA，从而满足对高质量DNA的需要。
Abstract: Cesium chloride (CsCl) centrifugation recovers DNA in large quantity, and recovery-column recovers DNA rapidly. The two methods’ advantages are combined to develop a CsCl centrifugation-column recovery method to produce DNAs in large-quantity, high-purification, and high-concentration. The study used 5.9 kb PCR-amplified DNA products as materials, ultra-centrifuged in a CsCl-EB solution at 75,000 rpm for 6 h, collected target DNAs by a syringe, and recovered target DNAs by a column to discard EB and CsCl. After CsCl centrifugation of 2000 μL PCR products, a 1-column recovery recovered DNAs in a 22.1 μg quantity and a high to 1.99 A260/A230 after a four-time elution. After CsCl centrifugation of 3000 μL of optimized PCR products, a 6-column recovery recovered DNAs in a high to 391 ng/μL concentration by a one-time elution and a high to 61.5 μg quantity by a four-time elution. In comparison, gel-recovery of 2000 μL PCR products recovered DNAs in a low to 9.6 μg quantity and a low to 0.29 A260/A230. Column-recovery of 2000 μL PCR products had many un-specific DNA bands. CsCl centrifugation-column recovery produces DNAs in large-quantity, high-purification, and high-concentration, and can fulfill demand for high quality of DNAs.
文章引用：邵玉强, 金家铖, 李雪刚, 刘亮伟. 大量–高纯–高浓度DNA的制备[J]. 微生物前沿, 2022, 11(2): 67-74. https://doi.org/10.12677/AMB.2022.112008

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