Restriction Enzymes BstNI / MvaI

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

MvaI (BstNI) (10 U/µL)

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
	To genotype the rs1801320, rs3218536, and rs861539 polymorphisms, 10 μL of each PCR product was digested with either 2 U of MvaI (BstNI) (Thermo Scientific, USA), 1 U of SexAI (New England Biolabs Inc., USA), or 0.5 units of NlaIII (New England Biolabs Inc., USA), respectively, for 16 h at 37°C. The genotypes were determined by running the digested products in 3% agarose gel with ethidium bromide (1 μL/mL) for UV visualization. The products for each genotype of the tested genes are shown in Table 3. Examples of the obtained restriction patterns are presented in Figure 1.

Protocol tips
To genotype the rs1801320, rs3218536, and rs861539 polymorphisms, 10 μL of each PCR product was digested with either 2 U of MvaI (BstNI) (Thermo Scientific, USA), 1 U of SexAI (New England Biolabs Inc., USA), or 0.5 units of NlaIII (New England Biolabs Inc., USA), respectively, for 16 h at 37°C. The genotypes were determined by running the digested products in 3% agarose gel with ethidium bromide (1 μL/mL) for UV visualization. The products for each genotype of the tested genes are shown in Table 3. Examples of the obtained restriction patterns are presented in Figure 1.

Protocol tips

To genotype the rs1801320, rs3218536, and rs861539 polymorphisms, 10 μL of each PCR product was digested with either 2 U of MvaI (BstNI) (Thermo Scientific, USA), 1 U of SexAI (New England Biolabs Inc., USA), or 0.5 units of NlaIII (New England Biolabs Inc., USA), respectively, for 16 h at 37°C. The genotypes were determined by running the digested products in 3% agarose gel with ethidium bromide (1 μL/mL) for UV visualization. The products for each genotype of the tested genes are shown in Table 3. Examples of the obtained restriction patterns are presented in Figure 1.

Manufacturer protocol Publication protocol 1 Relevant paper

FastDigest MvaI

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
	Digestion reactions were set up in 12 μl volume using 96 fmoles of duplex per reaction, with SUPERase•In™ RNase Inhibitor 1 U/μl, in the buffer recommended by the manufacturer. Enzymes were used at a maximum concentration compatible with glycerol content lower than 4.75%. MvaI, HindIII, PvuII (Fastdigest™ series) and BcnI enzymes were from Thermo Scientific™. AvaII, HinP1I and EcoRV were from New England Biolabs.

Protocol tips
Digestion reactions were set up in 12 μl volume using 96 fmoles of duplex per reaction, with SUPERase•In™ RNase Inhibitor 1 U/μl, in the buffer recommended by the manufacturer. Enzymes were used at a maximum concentration compatible with glycerol content lower than 4.75%. MvaI, HindIII, PvuII (Fastdigest™ series) and BcnI enzymes were from Thermo Scientific™. AvaII, HinP1I and EcoRV were from New England Biolabs.

Protocol tips

Digestion reactions were set up in 12 μl volume using 96 fmoles of duplex per reaction, with SUPERase•In™ RNase Inhibitor 1 U/μl, in the buffer recommended by the manufacturer. Enzymes were used at a maximum concentration compatible with glycerol content lower than 4.75%. MvaI, HindIII, PvuII (Fastdigest™ series) and BcnI enzymes were from Thermo Scientific™. AvaII, HinP1I and EcoRV were from New England Biolabs.

Manufacturer protocol Publication protocol 1 Relevant paper

BstNI NEB#R0168

New England BioLabs

Upstream tips	Protocol tips	Downstream tips
	Digestion of 157 bp PCR product of RAD51 (rs1801320) was dispatched with BstNI (NEB,USA) by incubating at 60° C for 4 hours which produced two fragments 86 bp and 71 bp in case of G/G allele while for C/C allele it produced only one fragment of 157 bp.

Protocol tips
Digestion of 157 bp PCR product of RAD51 (rs1801320) was dispatched with BstNI (NEB,USA) by incubating at 60° C for 4 hours which produced two fragments 86 bp and 71 bp in case of G/G allele while for C/C allele it produced only one fragment of 157 bp.

Publication protocol 1 Relevant paper

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