Construction of a New T-Vector: Nickase (Nt.BspQI)-Generated T-Vector Bearing a Reddish-Orange Indicator Gene.
10.1007/s13770-015-0118-z
- Author:
Ji Young CHOI
1
;
Chulman JO
;
Sangmee Ahn JO
Author Information
1. Department of Nanobiomedical Science & BK21 PLUS NBM Global Research Center for Regenerative Medicine, Dankook University, Cheonan, Korea. smahn@dankook.ac.kr
- Publication Type:Original Article
- Keywords:
T-vector;
DsRed gene;
Cloning;
Nickase;
Plasmid
- MeSH:
Clone Cells;
Cloning, Organism;
Deoxyribonuclease I*;
Methods;
Myostatin;
Plasmids;
Polymerase Chain Reaction
- From:
Tissue Engineering and Regenerative Medicine
2016;13(1):66-69
- CountryRepublic of Korea
- Language:English
-
Abstract:
T-vectors are widely used for cloning the polymerase chain reaction (PCR) products. However, the low conversion efficiency of a plasmid into the linear T-vector usually results in non-recombinants. Here, we designed a new plasmid pNBQ-T to easily select the recombinant colonies harboring PCR products. pNBQ-T plasmid, which contains a DsRed indicator gene between two Nt.BspQI restriction cassettes, each of which contains palindromic sequences susceptible to Nt.BspQI nickase (5′-GCTCTTCT^GAAGAGC-3′) at each end. Thus, this plasmid can be easily converted into T-vectors by a nickase (quadruple nicking), which results in two double strand breaks with 3′-thymidine overhangs. DsRed indicator gene, which is inserted between the restriction sites, helps identifying the PCR recombinants. Using this pNBQ-T plasmid the insertion efficiency of a PCR product was examined. We successfully identified white colony of the recombinants with the inserted myostatin promoter gene: the cloning efficiency was 93%. Therefore, this simple method utilizing pNBQ-T plasmid will serve as a useful and efficient technique for preparation of home-made T-vectors.
