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KMID : 0353320010250020089
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Oral Biology Research
2001 Volume.25 No. 2 p.89 ~ p.96
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Addition of 5 restriction enzymes site into multiple cloning sites of pCAT(R)3-Basic vector
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국중기/Kook JK
한진주/김명수/김홍중/박주철/Han JJ/Kim MJ/Kim HJ/Park JC
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Abstract
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To increase convenience and facilitate subcloning in contruction of chloramphenicol acetyl transferase (CAT) plasmid for transient expression assay, modification of the pCAT(R)3-Basic vector were performed. The procedures of this study were as follow: (1) The pCAT(R)3-Basic vector was digested with BamH I and Sal I and then the 5'-end overhanging were filled with Klenow Fragment and dNTPs. (2) The resulting blunt-end DNA fragment was self-ligated using T4 DNA ligase. (3) The logating mixture was transformed into E. coli DH5α. (4) The modified pCAT(R)3-Basic vector was named pCAT(R)3ΔB/S-Basic vector. (5) The pCAT(R)3ΔB/S-Basic vector was digested with Xba I and then the 5'-end overhanging were filled with Klenow Fragment and dNTPs. (6) The result modified pCAT(R)3ΔB/S-Basic vector was named pCAT(R)3b-Basic vector. (7) The multiple cloning site (from Kpn I to Xho I sites) of pCR2.1-TOPO cloned into the pCAT(R)3b-Basic vector. The resulting plasmid was named pCAT(R)3b-Basic vector. As the result, the new vector, pCAT(R)3b-CR-Basic vector has more five single cloning sites BamH I, Spe I, BstX I, EcoR V, and EcoR I-than the pCAT(R)3-Basic vector. This data indicate that the pCAT(R)3b-CR-Basic vector can be useful for construction of CAT plasmids in convenience and facility.
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