[Objective] The paper aimed to construct a new T vector based on plasmid pUC19 digested with Xcm Ⅰ. [Method] Two complementary oligonucleotide chains containing two Xcm Ⅰ sites were synthesized. After denaturation a...[Objective] The paper aimed to construct a new T vector based on plasmid pUC19 digested with Xcm Ⅰ. [Method] Two complementary oligonucleotide chains containing two Xcm Ⅰ sites were synthesized. After denaturation and renaturation,the adaptor was cloned into plasmid pUC19 between the Hind Ⅲ and BamH Ⅰ sites. The new plasmid,pUC19-HB-T vector,was digested with Xcm Ⅰ to derive a T-vector with 3′ end overhanging a T base. [Result] The constructed pUC19-HB-T vector was efficient in cloning PCR products,with an efficiency of 95% at least. [Conclusion] A new Xcm Ⅰ-based pUC19-HB-T vector was constructed,which could be applied to cloning of PCR products and other microbiology operations.展开更多
Two T vectors were generated by restriction endonuclease Xcm Ⅰ instead of Taq or other DNA polymerases to create the 3’T over hangs.A fragment of adenoviral genome position 10659-11865 was amplified by PCR and Xcm ...Two T vectors were generated by restriction endonuclease Xcm Ⅰ instead of Taq or other DNA polymerases to create the 3’T over hangs.A fragment of adenoviral genome position 10659-11865 was amplified by PCR and Xcm Ⅰ recognition sites were introduced to both ends of the PCR product.This fragment was cloned into the Sma Ⅰ site of pUC18 or the linearized pUC18 with its polylinker deleted.The recombinant plamids were cleaved with Xcm Ⅰ.the larger fragments generated which had 3’T over hangs at both ends were used as T vctors.Genes of rotavirus VP7 and the plasminogen k5 were successfully cloned into these two T vectors with recombination efficiency (recombinants/transformants×100%) of 100%,no blue/white clolny screening assay was needed.展开更多
文摘[Objective] The paper aimed to construct a new T vector based on plasmid pUC19 digested with Xcm Ⅰ. [Method] Two complementary oligonucleotide chains containing two Xcm Ⅰ sites were synthesized. After denaturation and renaturation,the adaptor was cloned into plasmid pUC19 between the Hind Ⅲ and BamH Ⅰ sites. The new plasmid,pUC19-HB-T vector,was digested with Xcm Ⅰ to derive a T-vector with 3′ end overhanging a T base. [Result] The constructed pUC19-HB-T vector was efficient in cloning PCR products,with an efficiency of 95% at least. [Conclusion] A new Xcm Ⅰ-based pUC19-HB-T vector was constructed,which could be applied to cloning of PCR products and other microbiology operations.
文摘Two T vectors were generated by restriction endonuclease Xcm Ⅰ instead of Taq or other DNA polymerases to create the 3’T over hangs.A fragment of adenoviral genome position 10659-11865 was amplified by PCR and Xcm Ⅰ recognition sites were introduced to both ends of the PCR product.This fragment was cloned into the Sma Ⅰ site of pUC18 or the linearized pUC18 with its polylinker deleted.The recombinant plamids were cleaved with Xcm Ⅰ.the larger fragments generated which had 3’T over hangs at both ends were used as T vctors.Genes of rotavirus VP7 and the plasminogen k5 were successfully cloned into these two T vectors with recombination efficiency (recombinants/transformants×100%) of 100%,no blue/white clolny screening assay was needed.
