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Table 5 SLiCE-mediated PCR-based site-directed mutagenesis

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

Mutation site

Number of coloniesc

Cloning efficiency

Mutation(%)d

Prx IIE C51Sa

22.0 ± 14.0

10/16

100

Prx IIE C76Sa

18.0 ± 7.5

15/16

100

G6PDH1 C97Sa

32.0 ± 4.6

9/16

93.3

G6PDH1 C105Sa

54.0 ± 13.5

9/16

88.9

Prx IIE C51Sb

197 ± 49.2

14/16

100

Prx IIE C76Sb

283 ± 21.2

15/16

100

G6PDH1 C97Sb

189 ± 39.0

6/16

100

G6PDH1 C105Sb

220 ± 30.0

9/16

100

  1. Target cysteine residues that are reduced by chloroplast thioredoxins were substituted with serine residues [26, 52]. The insert DNA fragments were prepared by PCR using PrimeSTAR Max DNA polymerase. pET23a and pET23d vectors were linearized using restriction enzymes NdeI and XhoI (for pET23a) and NcoI and XhoI (for pET23d). The SLiCE (JM109) reaction was performed for 60 min at 37 °C. aOne microliter of unpurified insert DNA fragments in 20 μL PCR solution was directly used for the SLiCE reaction. bPCR solution (20 μL) was purified using a Gel/PCR Extraction Kit (FastGene) and an equivalent volume to 1/20 of the PCR solution was used for the SLiCE reaction. cEach value of “number of colonies” is the mean ± standard deviation of three independent experiments. dThe ratio of mutants generated is given as the percentage of mutated clones (determined by DNA sequencing) among colony PCR-positive clones