Tissue Eng Regen Med.  2016 Feb;13(1):66-69. 10.1007/s13770-015-0118-z.

Construction of a New T-Vector: Nickase (Nt.BspQI)-Generated T-Vector Bearing a Reddish-Orange Indicator Gene

• Choi JY
• Jo C
• Jo SA

• KMID: 2360868
• DOI: http://doi.org/10.1007/s13770-015-0118-z

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"²-GCTCTTCT^GAAGAGC-3"²) 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"²-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.

Keyword

T-vector; DsRed gene; Cloning; Nickase; Plasmid

MeSH Terms

Clone Cells
Cloning, Organism
Deoxyribonuclease I*
Methods
Myostatin
Plasmids
Polymerase Chain Reaction
Deoxyribonuclease I
Myostatin