Table 2.

Efficiencies with which blunt-end PCR products were cloned into the blunt-end vectors carrying the E. coli-lethal ccdB gene.

Vector (Restriction enzyme for linearization)	Insert	Cloning efficiencies*a
pCRZero (EcoRV)	Prx IIE	21/24
pCRZero (EcoRV)	G6PDH1	22/24
pCRZeroT (SmaI)	G6PDH1	23/24

The pCRZero and pCRZeroT were digested with indicated restriction enzymes. The digested blunt-end vectors and, blunt-end PCR products (Prx IIE (0.6 kbp) or G6PDH1 (1.6 kbp) were purified by a Gel/PCR Extraction Kit. The blunt-end vectors (50 ng) and the blunt-end PCR products (Prx IIE 50 ng, G6PDH1 130 ng) were ligated with Quick ligase. Half the volume of ligation mixture was used to transform 50 μL of ECOS Competent E. coli DH5α chemically competent cells.

^*aThe cloning efficiencies are represented as “the number of clones with the confirmed correct length of insert DNA by colony-PCR/number of colonies subjected to colony PCR”.