Restriction Enzymes MseI / Tru1I

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 4 matching solutions for this experiment

Tru1I (MseI) (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

MseI NEB#R0525

New England BioLabs

Upstream tips	Protocol tips	Downstream tips
	genomic DNA from each sample was treated with NdeI, MseI (NEB, Ipswich, MA, USA), T4 DNA ligase (NEB), ATP (NEB), and MseI adapter at 37 °C. These restriction-ligation reaction solutions were diluted and mixed with dNTP, Taq DNA polymerase (NEB) and MseI primer containing barcode 1 for PCR reactions. The E.Z.N.A. Cycle Pure Kit (Omega, London, UK) were used to purify the PCR products. The purified PCR products were pooled and incubated at 37 °C with MseI, T4 DNA ligase, ATP, and Solexa adapter.	After incubation, the reaction products were then purified using a Quick Spin column (Qiagen, Venlo, Netherlands), and electrophoresed on a 2% agarose gel.

Protocol tips
genomic DNA from each sample was treated with NdeI, MseI (NEB, Ipswich, MA, USA), T4 DNA ligase (NEB), ATP (NEB), and MseI adapter at 37 °C. These restriction-ligation reaction solutions were diluted and mixed with dNTP, Taq DNA polymerase (NEB) and MseI primer containing barcode 1 for PCR reactions. The E.Z.N.A. Cycle Pure Kit (Omega, London, UK) were used to purify the PCR products. The purified PCR products were pooled and incubated at 37 °C with MseI, T4 DNA ligase, ATP, and Solexa adapter.
Downstream tips
After incubation, the reaction products were then purified using a Quick Spin column (Qiagen, Venlo, Netherlands), and electrophoresed on a 2% agarose gel.

Publication protocol 1 Relevant paper

MseI (RspRSII) restriction enzyme

Takara Bio Inc

Upstream tips	Protocol tips	Downstream tips
	PCR products were digested with the MseI (RspRSII) restriction enzymes (Takara) in a total volume of 20 μl at 60°C for 1 h based on the manufacturer’s instructions, with some modifications.	The digested fragments were separated in 2% agarose gels by electrophoresis in TBE buffer for approximately 45 min and visualized by staining with ethidium bromide.

Protocol tips
PCR products were digested with the MseI (RspRSII) restriction enzymes (Takara) in a total volume of 20 μl at 60°C for 1 h based on the manufacturer’s instructions, with some modifications.
Downstream tips
The digested fragments were separated in 2% agarose gels by electrophoresis in TBE buffer for approximately 45 min and visualized by staining with ethidium bromide.

Manufacturer protocol Publication protocol 1 Relevant paper

FastDigest Tru1I

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
	The nested PCR product was digested with TaaI and Tru1I FastDigest enzymes (Thermo Fisher Scientific Inc., Waltham, MA, USA) that produce diagnostic band patterns in agarose gel allowing to identify the two targeted species. With TaaI enzyme, brown trout DNA gives two bands of 205 and 272 bp, and with Tru1I, rainbow trout DNA gives a band of 66 bp.

Protocol tips
The nested PCR product was digested with TaaI and Tru1I FastDigest enzymes (Thermo Fisher Scientific Inc., Waltham, MA, USA) that produce diagnostic band patterns in agarose gel allowing to identify the two targeted species. With TaaI enzyme, brown trout DNA gives two bands of 205 and 272 bp, and with Tru1I, rainbow trout DNA gives a band of 66 bp.

Manufacturer protocol Publication protocol 1 Relevant paper

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