Table 5.

Blunting and blunt-end cloning of the dA-tailed DNA fragments in the blunt-end positive-selection system using the ccdB gene.

Vector (Restriction enzyme for linearization)	Blunting reaction	Number of colonies*a	Cloning efficiencies*b
pCRZeroT (SmaI)	None	11.3 ± 3.1	0/24
pCRZeroT (SmaI)	KOD DNA pol. 2 min	59.7 ± 22.0	14/24
pCRZeroT (SmaI)	KOD DNA pol. 30 min	84.7 ± 7.1	16/24

The pCRZeroT was linearized by blunt-end digestion with SmaI, and purified by a Gel/PCR Extraction Kit. The G6PDH1 (1.6 kbp) gene was amplified by KAPATaq EXtra DNA polymerase in a 50 μL reaction volume. The dNTPs (final 0.2 mM) and KOD DNA polymerase (1.25 U) were directly added to 50 μL of unpurified dA-tailed PCR-products. Blunting reactions were performed for 2 or 30 min at 72 °C. pCRZeroT (SmaI) (50 ng) and 2.5 μL of the 50 μL PCR reaction were then ligated. Three tenths of the ligation mixture were used to transform 30 μL of ECOS Competent E. coli DH5α chemically competent cells.

^*aThe number of colonies is the mean ± standard deviation of three independent experiments.

^*bThe 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”.