Boston University/TOPO Cloning
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Overview of TOPO Cloning: | Overview of TOPO Cloning: | ||
TOPO Cloning exploits the ligation activity of topoisomerase by providing an “activated”, linearized vector. Taq polymerase has a nontemplate-dependent terminal transferase activity that adds a single deoxyadenosine (A) to the 3’ ends of PCR products. The linearized vector supplied in the kit has single, overhanging 3’ deoxythimidine (T) residues. This allows PCR inserts to ligate efficiently with the vector. Ligation of the vector with a PCR product containing 3’ A-overhangs occurs spontaneously within 5 minutes at room temperature. After ligation, the pTrcHis-TOPO plasmid can be transformed into competent cells. | TOPO Cloning exploits the ligation activity of topoisomerase by providing an “activated”, linearized vector. Taq polymerase has a nontemplate-dependent terminal transferase activity that adds a single deoxyadenosine (A) to the 3’ ends of PCR products. The linearized vector supplied in the kit has single, overhanging 3’ deoxythimidine (T) residues. This allows PCR inserts to ligate efficiently with the vector. Ligation of the vector with a PCR product containing 3’ A-overhangs occurs spontaneously within 5 minutes at room temperature. After ligation, the pTrcHis-TOPO plasmid can be transformed into competent cells. | ||
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[[Boston_University | Back]] | [[Boston_University | Back]] | ||
Latest revision as of 16:21, 9 July 2007
TOPO cloning TOPO overview: The pTrcHis TOPO TA Expression Kit provides an efficient cloning strategy for the direct insertion of Taq polymerase-amplified PCR products into a plasmid vector for expression in E. coli. No ligase, post-PCR procedures, or PCR primers containing specific sequences are required. Once the PCR product of interest is TOPO Cloned into pTrcHis-TOPO, the construct is transformed into E. coli and expression is induced with isopropyl β-thiogalactoside(IPTG).
Overview of TOPO Cloning: TOPO Cloning exploits the ligation activity of topoisomerase by providing an “activated”, linearized vector. Taq polymerase has a nontemplate-dependent terminal transferase activity that adds a single deoxyadenosine (A) to the 3’ ends of PCR products. The linearized vector supplied in the kit has single, overhanging 3’ deoxythimidine (T) residues. This allows PCR inserts to ligate efficiently with the vector. Ligation of the vector with a PCR product containing 3’ A-overhangs occurs spontaneously within 5 minutes at room temperature. After ligation, the pTrcHis-TOPO plasmid can be transformed into competent cells.
