Restriction Enzymes HpaII / MspI

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 MspI

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
	15 µl of the resulting PCR product was digested with 2 µl of MspI enzyme (fast digest) and 3 µl enzyme buffer and then completed with nuclease-free water to a final volume of 30 µl, and incubated at 37°C for 30 min	Digested PCR products were electrophoresed using 3% agarose gel to distinguish between the 175 bp band produced by the digestion of the A allele and the 141 bp band produced by the digestion of the G allele. Heterozygous state yielded both 141 bp and 175 bp bands as previously described by Le Marchand et al. [19]

Protocol tips
15 µl of the resulting PCR product was digested with 2 µl of MspI enzyme (fast digest) and 3 µl enzyme buffer and then completed with nuclease-free water to a final volume of 30 µl, and incubated at 37°C for 30 min
Downstream tips
Digested PCR products were electrophoresed using 3% agarose gel to distinguish between the 175 bp band produced by the digestion of the A allele and the 141 bp band produced by the digestion of the G allele. Heterozygous state yielded both 141 bp and 175 bp bands as previously described by Le Marchand et al. [19]

Manufacturer protocol Publication protocol 1 Relevant paper

FastDigest HpaII

Thermo Fisher Scientific

Upstream tips	Protocol tips	Downstream tips
	DNA digestion reactions were set up with 200 ng DNA and 1 µL of each enzyme (AciI, MspI or HpaII FastDigest - purchased from Fermentas) in a total volume of 20 µL and incubated for 1 h at 37°C. Reaction products were loaded on 2% MetaPhor high resolution agarose (Lonza) gel with 0.4 µg/mL of Ethidium Bromide.

Protocol tips
DNA digestion reactions were set up with 200 ng DNA and 1 µL of each enzyme (AciI, MspI or HpaII FastDigest - purchased from Fermentas) in a total volume of 20 µL and incubated for 1 h at 37°C. Reaction products were loaded on 2% MetaPhor high resolution agarose (Lonza) gel with 0.4 µg/mL of Ethidium Bromide.

Manufacturer protocol Publication protocol 1 Relevant paper

HpaII R6311

Promega

Upstream tips	Protocol tips	Downstream tips
	Directional PCR and sequencing were used to confirm the correct orientation and sequences of chemokine gene inserts. P6-secretion signal-chemokine gene cassettes were then excised from pBR322 by ClaI restriction and inserted into pGK12, which was restricted with HpaII (Promega).

Protocol tips
Directional PCR and sequencing were used to confirm the correct orientation and sequences of chemokine gene inserts. P6-secretion signal-chemokine gene cassettes were then excised from pBR322 by ClaI restriction and inserted into pGK12, which was restricted with HpaII (Promega).

Manufacturer protocol Publication protocol 1 Relevant paper

HapII (HpaII, MspI) restriction enzyme

Takara Bio Inc

Upstream tips	Protocol tips	Downstream tips
	20 units EcoRI (Takara, P.R.China) and 20 units HpaII (Takara, P.R.China) were used to digest 200 ng genomic DNA in 20 µl of the reaction mixture at 37°C for 2 h.

Protocol tips
20 units EcoRI (Takara, P.R.China) and 20 units HpaII (Takara, P.R.China) were used to digest 200 ng genomic DNA in 20 µl of the reaction mixture at 37°C for 2 h.

Manufacturer protocol Publication protocol 1 Relevant paper

HpaII NEB#R0171

New England BioLabs

Upstream tips	Protocol tips	Downstream tips
	DNA (1 μg) was digested in NEB buffer 1 with 10 U of HpaII (NEB) in a 20-μl reaction volume for 3 hours at 37°C.

Protocol tips
DNA (1 μg) was digested in NEB buffer 1 with 10 U of HpaII (NEB) in a 20-μl reaction volume for 3 hours at 37°C.

Publication protocol 1 Relevant paper

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