利用XcmⅠ构建T载体-东南大学学报医学版

利用XcmⅠ构建T载体

单位：中国药科大学,生物技术中心,江苏,南京,210009

分类号：Q814

出版年·卷·期（页码）：2002·21·第四期（293-295）

摘要：

目的:构建能自我复制的T载体.方法:设计一对引物,引入XcmⅠ酶切位点,通过PCR方法用PET-28(a)+作模板扩增550bp,插入Pa载体后用限制性内切酶、PCR及DNA测序等方法作鉴定.结果:用XcmⅠ酶切可见540bp及3810bp双酶切条带,用此载体作模板可扩增出550bp片段,测序结果与预期序列一致.结论:带有两个XcmⅠ位点的环状载体被成功构建.

Objective To construct T-vector with characteristics of replicating itself.Methods By designing two primers on the site of XcmⅠ,550bp fragment was amplified by polymerase chain reaction(PCR) using template of PET28(a) +;the plasmid with inserted fragment was identified by methods of restrictive analysis,PCR and DNA sequencing.Results Two bands of 540bp and 3810bp appeared by XcmⅠ;550bp fragment can be amplified by PCR;and sequencing result was in accordance with the expected sequence.Conclusion A circular vector with two XcmⅠ sites has been successfully constructed.

参考文献：

[1] Marchuk D, DRUMM M, SAULINO A. Construction of T-vectors,a rapid and general system for direct cloning of unmodified PCR products. 1991. doi:10.1093/nar/19.5.1154
[2] Papp T, KIRCHNER S, DIENER U. Cloning of PCR fragments with a modified M13mp18 T-vector-polymerase chain reaction product cloning and DNA sequencing method. 1995. doi:10.1016/S0168-9525(0)89036-8
[3] Kawata Y, YANO S, KOJIMA H. Construction of a genomic DNA library by TA cloning, 1998
[4] Cha J, BISHAI W, CHANDRASEGARAN S. New vectors for direct cloning of PCR products. 1993. doi:10.1016/0378-1119(93)90498-R
[5] Ichihara Y, KUROSAWA Y. Construction of new T-vectors for direct cloning of PCR products. 1993. doi:10.1016/0378-1119(93)90361-6
[6] Harrison J, MOLLOY P L, Clark S J. Direct cloning of polymerase chain reaction products in an XcmⅠ T-vector. 1994. doi:10.1006/abio.1994.1033

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