Restriction Enzymes MboII

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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Found 3 matching solutions for this experiment

MboII NEB#R0148S

New England BioLabs

Upstream tips	Protocol tips	Downstream tips
	The RE-digestion and ligation solution contained 1 µL of Type IIS RE (MboII [R0148S], HphI [R0158S], BmrI [R0600S] or BciVI [R0596S] NEB), 0.3 µL of 1.25 μM L(G/A)-Boligo, 0.3 µL of 1.25 μM R(G/A)-Boligo, 3 µL of fragment (the concentration of fragment is recommended in section “Barcoding”), and 4.6 µL of Blunt/TA Ligase Master Mix. The RE-digestion mixture was incubated at 37 °C for 1 h, and two microliters of the solution was used as template in PCR to amplify barcoded fragment by using corresponding PS-modified Aoligos.

Protocol tips
The RE-digestion and ligation solution contained 1 µL of Type IIS RE (MboII [R0148S], HphI [R0158S], BmrI [R0600S] or BciVI [R0596S] NEB), 0.3 µL of 1.25 μM L(G/A)-Boligo, 0.3 µL of 1.25 μM R(G/A)-Boligo, 3 µL of fragment (the concentration of fragment is recommended in section “Barcoding”), and 4.6 µL of Blunt/TA Ligase Master Mix. The RE-digestion mixture was incubated at 37 °C for 1 h, and two microliters of the solution was used as template in PCR to amplify barcoded fragment by using corresponding PS-modified Aoligos.

Protocol tips

The RE-digestion and ligation solution contained 1 µL of Type IIS RE (MboII [R0148S], HphI [R0158S], BmrI [R0600S] or BciVI [R0596S] NEB), 0.3 µL of 1.25 μM L(G/A)-Boligo, 0.3 µL of 1.25 μM R(G/A)-Boligo, 3 µL of fragment (the concentration of fragment is recommended in section “Barcoding”), and 4.6 µL of Blunt/TA Ligase Master Mix. The RE-digestion mixture was incubated at 37 °C for 1 h, and two microliters of the solution was used as template in PCR to amplify barcoded fragment by using corresponding PS-modified Aoligos.

Publication protocol 1 Relevant paper

MboII 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

MboII (5 U/µL)

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
	Exon 1 (A/D, codon 52, rs5030737; A/B, codon 54, rs1800450 and A/C, codon 57, rs1800451) polymorphisms were investigated with the use of PCR–RFLP procedures, employing MluI, BshNI and MboII (all purchased from Thermo Fisher Scientific) enzymes, respectively66.

Protocol tips
Exon 1 (A/D, codon 52, rs5030737; A/B, codon 54, rs1800450 and A/C, codon 57, rs1800451) polymorphisms were investigated with the use of PCR–RFLP procedures, employing MluI, BshNI and MboII (all purchased from Thermo Fisher Scientific) enzymes, respectively66.

Manufacturer protocol Publication protocol 1 Relevant paper

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