Restriction Enzymes MnlI

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 2 matching solutions for this experiment

MnlI NEB#R0163

New England BioLabs

Upstream tips	Protocol tips	Downstream tips
	Ten microliters of PCR product were digested with 5 units of the MnlI restriction enzyme (NEB, Beverly, MA) following the manufacturer’s instructions in a final reaction volume of 30 µL.	The restriction fragments were separated by gel electrophoresis in 1X sodium borate buffer (Faster Better Media, Hunt Valley, MD) on 4.0% NuSieve GTG agarose gels containing ethidium bromide; the gel was photographed on a UV transilluminator. The gel patterns for each sample were scored according to MnlI fragment sizes (Table 1 and Fig 1A).

Protocol tips
Ten microliters of PCR product were digested with 5 units of the MnlI restriction enzyme (NEB, Beverly, MA) following the manufacturer’s instructions in a final reaction volume of 30 µL.
Downstream tips
The restriction fragments were separated by gel electrophoresis in 1X sodium borate buffer (Faster Better Media, Hunt Valley, MD) on 4.0% NuSieve GTG agarose gels containing ethidium bromide; the gel was photographed on a UV transilluminator. The gel patterns for each sample were scored according to MnlI fragment sizes (Table 1 and Fig 1A).

Publication protocol 1 Relevant paper

MnlI (10 U/µL)

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
	The product was digested using MnlI (Thermo Fisher Scientific, MA, USA)

Protocol tips
The product was digested using MnlI (Thermo Fisher Scientific, MA, USA)

Manufacturer protocol Publication protocol 1 Relevant paper

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