Construction of a Zero-background T-vector for Cloning PCR Products
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摘要: 为解决平末端加T法构建的T载体，在PCR产物克隆时连接效率低、阳性克隆筛选困难的问题，本研究在利用Taq DNA聚合酶末端转移酶活性对线性化平末端pBluescript SK（+）载体3'-端进行加T反应后，使用T4 DNA连接酶处理加T后pBS载体，使3'-端未加T平末端载体重新形成超螺旋状态，而3'-端加T载体则保持线性化状态，在琼脂糖电泳时切胶回收3 000 bp处DNA片段，成为pBS-T载体。使用构建的pBS-T载体分别克隆500 bp和1 000 bp PCR片段，pBS-T载体克隆效率达到100%，实现了对PCR产物的零背景克隆。Abstract: This experiment aimed to improve the shortcomings of low ligation efficiency and weak positive selection in cloning PCR products with T-vector.The approach called for an addition of T-overhang to a blunt-end plasmid. Firstly, a 3'-T overhang was attached to the blunt-end pBluescript SK (+) plasmid utilizing a terminal transferase, Taq DNA polymerase. Secondly, thepBS vector was ligated by T4 DNA ligase to form thesupercoiled blunt-end pBS plasmid without a 3'-T overhang, whereas the plasmidwith a 3'-T overhang remained linear in appearance. Finally, the DNA fragment of 3 000 bp, which was pBST-vector for cloning PCR products, could be purified byagarose gel electrophoresis followed by usinga gel extraction kit. The 500 bp and 1 000 bp DNA fragments were cloned into pBS T-vector with a100% cloning efficiency. It appeared that the newly developed procedures provided a valid zero-background T-vector for cloning PCR products.
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Key words:
- cloning PCR products /
- zero-background /
- T-vector
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图 1 pBS质粒酶切电泳
注：M为250 bp DNA Ladder；1为未酶切pBS质粒；2为酶切后pBS质粒。
Figure 1. Agarose gel electrophoretic patterns of pBS plasmid digested by Eco RV
图 2 pBS载体加T效率检测和菌落PCR验证
注：M为DL2000 DNA Marker；1~5为白斑菌落PCR验证结果；6~10为蓝斑菌落PCR验证结果。
Figure 2. Cloning efficiency of pBS vector and identification by colony PCR
图 3 回收后pBS-T载体电泳
注：M为250 bp DNA Ladder；1为T4 DNA连接酶处理后pBS载体；2为切胶回收后pBS-T载体。
Figure 3. Agarose gel electrophoretic patterns of purified pBST-vector
图 4 pBS-T载体克隆效率检测和菌落PCR验证
注：M为DL2000 DNA Marker；1~10为白斑菌落PCR验证结果。
Figure 4. Cloning efficiency of 500 bp DNA fragments inserted into pBST-vector and identification by colony PCR
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