Skip to main content

Table 4 Effect of PCR fragment purification method on SLiCE efficiency

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

Purification method

Number of coloniesf

Cloning efficiency

Prx IIE

G6PDH1

Prx IIE

G6PDH1

Nonea

86.3 ± 2.3

76.0 ± 4.0

15/18

17/18

EtOH pptb

546 ± 102

514 ± 68.7

18/18

17/18

ExoSAP-ITc

481 ± 117

507 ± 105

18/18

18/18

Columnd

7,170 ± 806

4,660 ± 1,400

18/18

18/18

Agarose gele

11,900 ± 1,750

2,830 ± 100

18/18

18/18

  1. The insert DNA fragments were amplified using 19-bp overlap primers and KOD DNA polymerase [51] (for Prx IIE gene) and PrimeSTAR Max DNA polymerase (for G6PDH1). The linearized vector DNA was prepared by PCR. The PCR solutions were treated with DpnI. The SLiCE (JM109) reaction was performed for 10 min at 37 °C. aUnpurified insert DNA was directly used for the SLiCE reaction. bInsert DNA was precipitated by ethanol. cPCR solution was treated with ExoSAP-IT (Affymetrix). dInsert DNA was purified from the PCR solution using a Gel/PCR Extraction Kit (FastGene). eInsert DNA was purified by agarose gel electrophoresis and using a Gel/PCR Extraction Kit (FastGene). An equivalent volume to 1/20 of the PCR solution was used for the SLiCE reaction. fEach value of “number of colonies” is the mean ± standard deviation of three independent experiments