Restriction Enzymes MluI

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 5 matching solutions for this experiment

FastDigest MluI

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
	Genomic DNA (1 μg) was digested for 20 min at 37°C with 1 μl of FastDigest MluI (Thermo Fisher Scientific), 1 μl of FastDigest SbfI (Thermo Fisher Scientific), and 2 μl 10× FastDigest buffer (Thermo Fisher Scientific) in a total volume of 20 μl.	The digestion was purified using AMPure XP beads (Beckman Coulter), with a ratio of 1.8:1 to the sample, followed by resuspension in 50 μl of ddH2O. Then, restriction‐associated double‐stranded Y adapters were ligated to the purified fragments in a 60 μl reaction containing 10 μM MluI adapter, 10 μM SbfI adapter, 5U T4 DNA ligase (Fermentas Inc.), 100 mM ATP, and 1× T4 ligation buffer.

Protocol tips
Genomic DNA (1 μg) was digested for 20 min at 37°C with 1 μl of FastDigest MluI (Thermo Fisher Scientific), 1 μl of FastDigest SbfI (Thermo Fisher Scientific), and 2 μl 10× FastDigest buffer (Thermo Fisher Scientific) in a total volume of 20 μl.
Downstream tips
The digestion was purified using AMPure XP beads (Beckman Coulter), with a ratio of 1.8:1 to the sample, followed by resuspension in 50 μl of ddH2O. Then, restriction‐associated double‐stranded Y adapters were ligated to the purified fragments in a 60 μl reaction containing 10 μM MluI adapter, 10 μM SbfI adapter, 5U T4 DNA ligase (Fermentas Inc.), 100 mM ATP, and 1× T4 ligation buffer.

Manufacturer protocol Publication protocol 1 Relevant paper

MluI NEB#R0198

New England BioLabs

Upstream tips	Protocol tips	Downstream tips
	Restriction enzymes MluI (R0198), SalI-HF (R3138) and XhoI (R0146) were from NEB. Buffer 3.1 (B7203; final concentration: 100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2, 100 μg/ml BSA, pH 7.9) was used for MluI-, SalI- and MluI-, XhoI-double-digests. 1 μg of plasmid DNA or PCR product was incubated with 5 U of each restriction enzyme in a final volume of 30 μl for 1 h at 37°C. DNA fragments were separated on 0.8% Agarose (Sigma) gels and purified using the QIAquick gel purification Kit (Qiagen).

Protocol tips
Restriction enzymes MluI (R0198), SalI-HF (R3138) and XhoI (R0146) were from NEB. Buffer 3.1 (B7203; final concentration: 100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2, 100 μg/ml BSA, pH 7.9) was used for MluI-, SalI- and MluI-, XhoI-double-digests. 1 μg of plasmid DNA or PCR product was incubated with 5 U of each restriction enzyme in a final volume of 30 μl for 1 h at 37°C. DNA fragments were separated on 0.8% Agarose (Sigma) gels and purified using the QIAquick gel purification Kit (Qiagen).

Protocol tips

Restriction enzymes MluI (R0198), SalI-HF (R3138) and XhoI (R0146) were from NEB. Buffer 3.1 (B7203; final concentration: 100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2, 100 μg/ml BSA, pH 7.9) was used for MluI-, SalI- and MluI-, XhoI-double-digests. 1 μg of plasmid DNA or PCR product was incubated with 5 U of each restriction enzyme in a final volume of 30 μl for 1 h at 37°C. DNA fragments were separated on 0.8% Agarose (Sigma) gels and purified using the QIAquick gel purification Kit (Qiagen).

Publication protocol 1 Relevant paper

MluI restriction enzyme

Takara Bio Inc

Upstream tips	Protocol tips	Downstream tips
	Plasmid pAsi, the infectious cDNA clone of Asia1 FMDV Asia1/YS/CHA/05, was digested with MluI and EcoRV or SmaI and MluI (TaKaRa, Dalian, China). Positive plasmids bearing the desired mutation(s) in 3Dpol were digested with MluI and EcoRV or SmaI and MluI and reintroduced into pAsi that had been digested previously with the same restriction endonucleases. The recombinant plasmids were used for in vitro transcription and transfection.

Protocol tips
Plasmid pAsi, the infectious cDNA clone of Asia1 FMDV Asia1/YS/CHA/05, was digested with MluI and EcoRV or SmaI and MluI (TaKaRa, Dalian, China). Positive plasmids bearing the desired mutation(s) in 3Dpol were digested with MluI and EcoRV or SmaI and MluI and reintroduced into pAsi that had been digested previously with the same restriction endonucleases. The recombinant plasmids were used for in vitro transcription and transfection.

Protocol tips

Plasmid pAsi, the infectious cDNA clone of Asia1 FMDV Asia1/YS/CHA/05, was digested with MluI and EcoRV or SmaI and MluI (TaKaRa, Dalian, China).
Positive plasmids bearing the desired mutation(s) in 3Dpol were digested with MluI and EcoRV or SmaI and MluI and reintroduced into pAsi that had been digested previously with the same restriction endonucleases. The recombinant plasmids were used for in vitro transcription and transfection.

Manufacturer protocol Publication protocol 1 Relevant paper

MluI-HF®

New England BioLabs

Upstream tips	Protocol tips	Downstream tips
	10 micrograms of each PCR product was digested with MluI-HF and SphI-HF and gel extracted with the QIAquick Gel Extraction kit (Qiagen, Hilden, Germany). 20 micrograms of the first-step cloning product was digested with MluI-HF and SphI-HF (New England Biolabs, Ipswich, MA), treated with Shrimp Alkaline Phosphatase, and purified with the QIAquick PCR purification kit.

Protocol tips
10 micrograms of each PCR product was digested with MluI-HF and SphI-HF and gel extracted with the QIAquick Gel Extraction kit (Qiagen, Hilden, Germany). 20 micrograms of the first-step cloning product was digested with MluI-HF and SphI-HF (New England Biolabs, Ipswich, MA), treated with Shrimp Alkaline Phosphatase, and purified with the QIAquick PCR purification kit.

Publication protocol 1 Relevant paper

MluI R6381

Promega

Upstream tips	Protocol tips	Downstream tips
	The wild-type and mutant CLOCK 3×RORE oligos were digested with XhoI and MluI (Promega). The pTL-Luciferase vector was also digested with XhoI and MluI (Promega).	All fragments were gel purified and ligated using T4 DNA Ligase (Promega). The constructs were verified by sequencing.

Protocol tips
The wild-type and mutant CLOCK 3×RORE oligos were digested with XhoI and MluI (Promega). The pTL-Luciferase vector was also digested with XhoI and MluI (Promega).
Downstream tips
All fragments were gel purified and ligated using T4 DNA Ligase (Promega). The constructs were verified by sequencing.

Manufacturer protocol Publication protocol 1 Relevant paper

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