DpnI restriction enzyme

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	The TTcDR reaction was performed using plasmids containing each DNA fragment (original, clone 6, clone 6-wt-loxP) and an ampicillin resistance gene in the presence or absence of 30 mU/μl Cre recombinase for 0 or 16 h at 30 °C. The reaction mixture was then diluted 20-fold and treated with 500 mU/μl DpnI (TaKaRa) at 37 °C for 2 h to degrade the initial plasmid DNA. The mixtures were purified and diluted 2.5-fold using a DNA column (PureLink PCR micro Kit). The purified DNA was transformed into an E. coli strain (DH5α) by a chemical method and spread onto a Luria-Bertani agar plate containing 50 μg/ml ampicillin. After 16 h of incubation at 37 °C, the number of colonies was counted.

Protocol tips

The TTcDR reaction was performed using plasmids containing each DNA fragment (original, clone 6, clone 6-wt-loxP) and an ampicillin resistance gene in the presence or absence of 30 mU/μl Cre recombinase for 0 or 16 h at 30 °C. The reaction mixture was then diluted 20-fold and treated with 500 mU/μl DpnI (TaKaRa) at 37 °C for 2 h to degrade the initial plasmid DNA. The mixtures were purified and diluted 2.5-fold using a DNA column (PureLink PCR micro Kit). The purified DNA was transformed into an E. coli strain (DH5α) by a chemical method and spread onto a Luria-Bertani agar plate containing 50 μg/ml ampicillin. After 16 h of incubation at 37 °C, the number of colonies was counted.

Publication protocol

The TTcDR reaction was performed using plasmids containing each DNA fragment (original, clone 6, clone 6-wt-loxP) and an ampicillin resistance gene in the presence or absence of 30 mU/μl Cre recombinase for 0 or 16 h at 30 °C. The reaction mixture was then diluted 20-fold and treated with 500 mU/μl DpnI (TaKaRa) at 37 °C for 2 h to degrade the initial plasmid DNA. The mixtures were purified and diluted 2.5-fold using a DNA column (PureLink PCR micro Kit). The purified DNA was transformed into an E. coli strain (DH5α) by a chemical method and spread onto a Luria-Bertani agar plate containing 50 μg/ml ampicillin. After 16 h of incubation at 37 °C, the number of colonies was counted.

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