Table 3.

Direct PCR cloning into non-digested vectors using the Golden Gate reaction.

vector	Restriction enzyme (Buffer)	Insert	White colonies (%)*a	Cloning efficiencies*b
pCRZeroT	XcmI/XmaI (CutSmart)	G6PDH1	100.0 ± 0.0	21/24
pCRZeroT	SmaI (CutSmart)	G6PDH1	—	24/24
pCRZero	EcoRV (CutSmart)	Prx IIE	—	23/24
pZErO2.1	EcoRV (CutSmart)	Prx IIE	—	24/24

The PCR products (G6PDH1; 1.6 kbp or Prx IIE; 0.6 kbp) were purified by a Gel/PCR Extraction Kit. The pCRZeroT, pCRZero, or pZErO2.1 (Non-digested, 50 ng) and the purified blunt-end PCR products (G6PDH1 130 ng or Prx IIE 50 ng) were ligated with Quick ligase and each restriction enzyme in the Golden Gate reaction. Half of the ligation mixture volume was used to transform 50 μL of ECOS Competent E. coli DH5α chemically competent cells. Plasmid pZErO2.1 was used as a control for commercially available vectors, and positive clones in pZErO2.1 were screened on an LB agar plate containing kanamycin (50 μg/mL).

^*aThe fraction of white colonies as a percentage of total colonies was calculated and expressed as mean percentage ± 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”.