A simple and efficient seamless DNA cloning method using SLiCE from Escherichia coli laboratory strains and its application to SLiP site-directed mutagenesis

Table 3 The SLiCE cloning efficiencies of linearized vectors prepared by digestion with different restriction enzymes

Restriction enzymes	Flanking heterologous length (bp)	Number of colonies^a		Cloning efficiency		Cloning accuracy (%)
Restriction enzymes	Flanking heterologous length (bp)	Prx IIE	G6PDH1	Prx IIE	G6PDH1	Prx IIE	G6PDH1
NdeI - XhoI	0 + 0	9,680 ± 651	-	18/18	-	100	-
NcoI - XhoI	0 + 0	-	8,470 ± 2,180	-	17/18	-	94.1
BamHI	40 + 40	926 ± 62.0	576 ± 17.6	1/18	5/18	100	100
NdeI - BamHI	0 + 40	164 ± 20.8	-	12/18	-	75.0	-
NcoI - BamHI	0 + 40	-	195 ± 83.3	-	4/18	-	100
BamHI - XhoI	40 + 0	857 ± 298	960 ± 71.3	9/18	12/18	88.9	83.3

The insert DNA fragment was prepared by PCR. The linearized pET23a or pET23d vectors were prepared by digestion with restriction enzymes. The multiple cloning site of pET23a (or pET23d) are displayed in Additional file 1: Figure S1. The SLiCE (JM109) reaction was performed for 60 min at 37 °C with 1:1 and 3:1 molar ratios of insert to vector for Prx IIE and G6PDH1, respectively. ^aEach value of “number of colonies” is the mean ± standard deviation of three independent experiments

ISSN: 1472-6750