Restriction Enzymes DpnI

A restriction enzyme or restriction endonuclease is defined as a protein that recognizes a specific, short nucleotide sequence and cuts the DNA only at or near that site, known as restriction site or target sequence. The four most common types of restriction enzymes include: Type I (cleaves at sites remote from a recognition site), Type II (cleaves within or at short specific distances from a recognition site), Type III (cleave at sites a short distance from a recognition site), and Type IV (targets modified DNA- methylated, hydroxymethylated and glucosyl-hydroxymethylated DNA). The most common challenges with restriction digest include- 1. inactivation of the enzyme, 2. incomplete or no digestion, and 3. unexpected cleavage. The enzyme should always be stored at -20C and multiple freeze-thaw cycles should be avoided in order to maintain optimal activity. Always use a control DNA digestion with the enzyme to ensure adequate activity (to avoid interference due to high glycerol in the enzyme). For complete digestion, make sure that the enzyme volume is 1/10th of the total reaction volume, the optimal temperature is constantly maintained throughout the reaction, the total reaction time is appropriately calculated based on the amount of DNA to be digested, appropriate buffers should be used to ensure maximal enzymatic activity, and in case of a double digest, make sure that the two restriction sites are far enough so that the activity of one enzyme cannot interfere with the activity of the other. Star activity (or off-target cleavage) and incomplete cleavage are potential challenges which may occur due to suboptimal enzymatic conditions or inappropriate enzyme storage. To avoid these, follow the recommended guidelines for storage and reactions, and always check for the efficacy of digestion along with purification of digested products on an agarose gel.

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DpnI restriction enzyme

Takara Bio Inc

Upstream tips	Protocol tips	Downstream 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.

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.

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.

Manufacturer protocol Publication protocol 1 Relevant paper

DpnI (10 U/µL)

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
	We added 0.8 μl 10 x FD buffer and 0.2 μl DpnI (Thermo Scientific) to the final PCR product, and the mix was directly transformed into E. coli strain DH5α. Three single colonies were cultured, and plasmids extracted using the GeneJET Plasmid Miniprep Kit (Thermo Scientific), followed by Sanger-sequencing to confirm sgRNA integration. sgRNA sequences and vectors are listed in Additional file 5: Table S3.

Protocol tips
We added 0.8 μl 10 x FD buffer and 0.2 μl DpnI (Thermo Scientific) to the final PCR product, and the mix was directly transformed into E. coli strain DH5α. Three single colonies were cultured, and plasmids extracted using the GeneJET Plasmid Miniprep Kit (Thermo Scientific), followed by Sanger-sequencing to confirm sgRNA integration. sgRNA sequences and vectors are listed in Additional file 5: Table S3.

Protocol tips

We added 0.8 μl 10 x FD buffer and 0.2 μl DpnI (Thermo Scientific) to the final PCR product, and the mix was directly transformed into E. coli strain DH5α. Three single colonies were cultured, and plasmids extracted using the GeneJET Plasmid Miniprep Kit (Thermo Scientific), followed by Sanger-sequencing to confirm sgRNA integration. sgRNA sequences and vectors are listed in Additional file 5: Table S3.

Manufacturer protocol Publication protocol 1 Relevant paper

DpnI NEB#R0176

New England BioLabs

Upstream tips	Protocol tips	Downstream tips
	The PCR reaction was followed by an overnight digestion at 37°C through addition of 2 μl (4000 units) of DpnI (NEB, Hitchin, UK). 5 μl of the PCR reaction were subsequently transformed in E. coli strain Top10. Ten colonies per plate were isolated and analysed via restriction digest and sequencing (GATC Biotech, Konstanz, Germany).

Protocol tips
The PCR reaction was followed by an overnight digestion at 37°C through addition of 2 μl (4000 units) of DpnI (NEB, Hitchin, UK). 5 μl of the PCR reaction were subsequently transformed in E. coli strain Top10. Ten colonies per plate were isolated and analysed via restriction digest and sequencing (GATC Biotech, Konstanz, Germany).

Publication protocol 1 Relevant paper

FastDigest DpnI

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
	Point mutations were generated by site-directed mutagenesis with FastDigest DpnI (Fermentas, FD1704) using mutagenic primers for PCR, and the plasmids encoding the wild-type protein were used as the template. All constructs were confirmed by DNA sequencing.

Protocol tips
Point mutations were generated by site-directed mutagenesis with FastDigest DpnI (Fermentas, FD1704) using mutagenic primers for PCR, and the plasmids encoding the wild-type protein were used as the template. All constructs were confirmed by DNA sequencing.

Manufacturer protocol Publication protocol 1 Relevant paper

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