| 摘要: |
| 微绿球藻(Nannochloropsis oculata)DNA被提取纯化并经超声波处理后,将所得大小在1.6～3 kb之间的DNA片段用T4 DNA聚合酶补平,再与SmaI酶切消化并经去磷酸化处理的质粒载体pUC18连接,转化至DH10B大肠杆菌(Escherichia coli)的感受态细胞中。建立的DNA质粒文库容量含2×104个克隆,其中重组子占90%。随机挑选白色菌落并培养,抽提的质粒经XbaI和SacI 双酶切鉴定,显示重组的质粒中均含有大小不等的DNA插入片段。 |
| 关键词: 微绿球藻(Nannochloropsis oculata) 超声处理 质粒载体pUC18 DNA文库 |
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| 基金项目:国家转基因植物研究与产业化开发专项(JY03-B-20);上海市属高校自然科学研究资助项目(01H04) |
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| DNA library of Nannochloropsis oculata constructed with plasmid vector pUC18 |
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| Abstract: |
| The genomic DNA isolated and purified from Nannochloropsis oculata was sheared after a treatment with an ultrasonic processor. The treated DNA was blunt-ended with T4 DNA polymerase and then was collected from 1.6 kb to 3 kb in size. The target blunt-ended DNA was cloned into plasmid vector pUC18 which was previously digested with Sma I and dephosphorylated with calf alkaline phosphatase. The recombinant plasmid was transformed into Escherichia coli DH10B competent cells and the genomic library was constructed. There were about 2×104 clones in the genomic library of N. oculata, and the percentage of recombinants was about 90%. It was confirmed that all randomly selected plasmids of white colonies contained target DNA inserts from 1.6 kb to 3 kb in size after restriction mapping with Xba I and Sac I endonucleases. |
| Key words: Nannochloropsis oculata ultrasonication plasmid vector pUC18 genomic library |
