Restriction Enzymes MboI

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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MboI (10 U/µL)

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
	The 16S rDNA amplicons were digested with 3 restriction enzymes – AluI, MseI and MboI (Thermo Scientific, USA), which were selected on the basis of in silico analysis using CLC Main Workbench software (Qiagen) and the 16S rDNA nucleotide sequences of the Lactobacillus strains, deposited in GenBank. Ten μl of PCR product was digested in 12.7 μl of restriction enzyme buffer containing 0.7 μl of enzyme (initial concentration of each restriction enzyme 10 U/μl) and left to react at 65°C (for MseI) or at 37°C (for AluI and MboI) for 4 h. DNA electrophoresis and analysis of restriction profiles were carried out as described in previous work [13].

Protocol tips
The 16S rDNA amplicons were digested with 3 restriction enzymes – AluI, MseI and MboI (Thermo Scientific, USA), which were selected on the basis of in silico analysis using CLC Main Workbench software (Qiagen) and the 16S rDNA nucleotide sequences of the Lactobacillus strains, deposited in GenBank. Ten μl of PCR product was digested in 12.7 μl of restriction enzyme buffer containing 0.7 μl of enzyme (initial concentration of each restriction enzyme 10 U/μl) and left to react at 65°C (for MseI) or at 37°C (for AluI and MboI) for 4 h. DNA electrophoresis and analysis of restriction profiles were carried out as described in previous work [13].

Protocol tips

The 16S rDNA amplicons were digested with 3 restriction enzymes – AluI, MseI and MboI (Thermo Scientific, USA), which were selected on the basis of in silico analysis using CLC Main Workbench software (Qiagen) and the 16S rDNA nucleotide sequences of the Lactobacillus strains, deposited in GenBank.

Ten μl of PCR product was digested in 12.7 μl of restriction enzyme buffer containing 0.7 μl of enzyme (initial concentration of each restriction enzyme 10 U/μl) and left to react at 65°C (for MseI) or at 37°C (for AluI and MboI) for 4 h. DNA electrophoresis and analysis of restriction profiles were carried out as described in previous work [13].

Manufacturer protocol Publication protocol 1 Relevant paper

FastDigest MboI

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
	2200 bp bands amplified from gDNA of five +B and five 0B mice were removed from the agarose gels, purified with QIAquick Gel Extraction Kit (Qiagen) and then digested with twelve restriction endonucleases (RE): ApaI, HindIII, Bsp120I, MfeI, HpaI, MspI, MboI, AluI, MseI, Ecl136II, HaeIII and SacI (FastDigest, Thermo Scientific, Waltham, MA USA) according to the manufacturer’s instructions. Digested DNA fragments were separated by agarose gel electrophoresis, visualized and photo-documented as above.

Protocol tips
2200 bp bands amplified from gDNA of five +B and five 0B mice were removed from the agarose gels, purified with QIAquick Gel Extraction Kit (Qiagen) and then digested with twelve restriction endonucleases (RE): ApaI, HindIII, Bsp120I, MfeI, HpaI, MspI, MboI, AluI, MseI, Ecl136II, HaeIII and SacI (FastDigest, Thermo Scientific, Waltham, MA USA) according to the manufacturer’s instructions. Digested DNA fragments were separated by agarose gel electrophoresis, visualized and photo-documented as above.

Protocol tips

2200 bp bands amplified from gDNA of five +B and five 0B mice were removed from the agarose gels, purified with QIAquick Gel Extraction Kit (Qiagen) and then digested with twelve restriction endonucleases (RE): ApaI, HindIII, Bsp120I, MfeI, HpaI, MspI, MboI, AluI, MseI, Ecl136II, HaeIII and SacI (FastDigest, Thermo Scientific, Waltham, MA USA) according to the manufacturer’s instructions. Digested DNA fragments were separated by agarose gel electrophoresis, visualized and photo-documented as above.

Manufacturer protocol Publication protocol 1 Relevant paper

MboI (Sau3AI) restriction enzyme

Takara Bio Inc

Upstream tips	Protocol tips	Downstream tips
	Each DNA was incubated with 10 U restriction enzyme [MboII (Takara), AlwI (New England Biolabs, Beverly, USA) or MboI (Takara)] in 10 μl of a buffer {MboII buffer [10 mM Tris–HCl (pH 7.5), 10 mM MgCl2 and 1 mM DTT], AlwI buffer [20 mM Tris–HCl (pH 7.9), 10 mM MgCl2, 1 mM DTT and 50 mM KCl] and MboI buffer [20 mM Tris–HCl (pH 8.5), 10 mM MgCl2, 1 mM DTT and 100 mM KCl]} at 37°C for 1 h. The surrounding sequences of the recognition sequences are shown in Figure 1

Protocol tips
Each DNA was incubated with 10 U restriction enzyme [MboII (Takara), AlwI (New England Biolabs, Beverly, USA) or MboI (Takara)] in 10 μl of a buffer {MboII buffer [10 mM Tris–HCl (pH 7.5), 10 mM MgCl2 and 1 mM DTT], AlwI buffer [20 mM Tris–HCl (pH 7.9), 10 mM MgCl2, 1 mM DTT and 50 mM KCl] and MboI buffer [20 mM Tris–HCl (pH 8.5), 10 mM MgCl2, 1 mM DTT and 100 mM KCl]} at 37°C for 1 h. The surrounding sequences of the recognition sequences are shown in Figure 1

Protocol tips

Each DNA was incubated with 10 U restriction enzyme [MboII (Takara), AlwI (New England Biolabs, Beverly, USA) or MboI (Takara)] in 10 μl of a buffer {MboII buffer [10 mM Tris–HCl (pH 7.5), 10 mM MgCl2 and 1 mM DTT], AlwI buffer [20 mM Tris–HCl (pH 7.9), 10 mM MgCl2, 1 mM DTT and 50 mM KCl] and MboI buffer [20 mM Tris–HCl (pH 8.5), 10 mM MgCl2, 1 mM DTT and 100 mM KCl]} at 37°C for 1 h. The surrounding sequences of the recognition sequences are shown in Figure 1

Manufacturer protocol Publication protocol 1 Relevant paper

MboI NEB#R0147

New England BioLabs

Upstream tips	Protocol tips	Downstream tips
	Chromatin was then digested by adding 50 μl of NEBuffer 2 10X and 350 units of MboI (NEB R0147M) at 37 °C for 2 h while rotating at 950 rpm. MboI was inactivated by incubating the samples 20 min at 62 °C.

Protocol tips
Chromatin was then digested by adding 50 μl of NEBuffer 2 10X and 350 units of MboI (NEB R0147M) at 37 °C for 2 h while rotating at 950 rpm. MboI was inactivated by incubating the samples 20 min at 62 °C.

Publication protocol 1 Relevant paper

MboI R6711

Promega

Upstream tips	Protocol tips	Downstream tips
	Genomic DNA was extracted from the pterygiophore which preserved by 70% ethanol, and then digested by restriction enzyme MboI (Promega) for the fragments were distributed at the 400– 900 bp.

Protocol tips
Genomic DNA was extracted from the pterygiophore which preserved by 70% ethanol, and then digested by restriction enzyme MboI (Promega) for the fragments were distributed at the 400– 900 bp.

Manufacturer protocol Publication protocol 1 Relevant paper

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