Restriction Enzymes MfeI / MunI

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

FastDigest MunI

Thermo Fisher Scientific

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.
MfeI-HF®

New England BioLabs

Protocol tips
Purified DNA was digested with 25 units/µg MfeI-HF (New England Biolabs) in 50 µl µg−1 reaction volumes for 16 h at 37 °C according to the manufacturer’s instructions. After 16 h, an additional 5 units µg−1 enzyme was added to the reactions and incubation was continued an additional 2 h at 37 °C.
Downstream tips
Digested DNA was purified by phenol:chloroform: isoamyl alcohol extraction and ethanol precipitation as described above
MunI (MfeI) (10 U/µL)

Thermo Fisher Scientific

Protocol tips
The mutation in the fifth exon of the NBN gene (p.I171V) was assessed by restriction fragment length polymorphism (RFLP) analysis of polymerase chain reaction (PCR) products, using MunI (MfeI) restriction enzyme (Fermentas, Thermo Fisher Scientific, Inc., Pittsburgh, PA, USA).
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