Restriction Enzymes HinfI

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

HinfI (10 U/µL)

Thermo Fisher Scientific

Protocol tips
For 2D agarose gel analysis, 10μg DNA was digested overnight at 37°C with 10U HinfI (Thermo Fisher), 10U RsaI (Thermo Fisher) and 2μg/mL RNase A (Takara). The reaction was terminated with EDTA and analyzed by 2D agarose gel electrophoresis. 30U RecJf (New England Biolabs) was added for removing 5' single-stranded DNA.

For internal gaps/nicks analysis, 5μg genomic DNA was digested overnight at 37°C with 5U HinfI (Thermo Fisher), 5U RsaI (Thermo Fisher) and 1μg/mL Ribonuclease A (RNase A, Takara) and purified with QIAquick PCR Purification kit (Qiagen). Purified DNA were digested with or without 200U Exonuclease III (New England Biolabs) overnight at 37°C, and then subjected to 0.7% agarose gel electrophoresis and in-gel hybridization.
Protocol tips
According to the SNP mutation site gene, we selected the restriction enzyme HinfI (Takara Bio, Inc.) for the treatment of PCR amplified fragments, to carry on the identification of the gene. All of the 180 GDM and 210 in the control group PCR amplified fragments were by HinfI. The reaction system contained Takara HinfI 1 μl, 10X HinfI buffer 2 μl, PCR template 7-8 μl, add ultrapure water to 20 μl, kept in 37°C water for 8 h. The fragments digested by HinfI were electrophoresed in a 3% agarose gel. Genotypes were determined by the results of various bands though UV light (c150; Azure Biosystems, Inc.).
HinfI NEB#R0155

New England BioLabs

Protocol tips
RFLP was carried out in 30 μl reaction containing 0.5 μg (10-15 μl) PCR product, RE buffer, 2U of HaeIII / HinfI / XhoI restriction enzyme from NEB [9, 12]. Digestion was carried out for 1 h 30 min. Digests were electrophoresed on 1% agarose gel and band patterns were analyzed by visual comparison.
Protocol tips
Aliquots of 10 μL of each 275 bp amplified product were digested with restriction endonucleases as recommended by the manufacturer. Three restriction enzymes, DdeI, HaeIII and HinfI (Promega, Madison, WI, USA), were used in separate reactions.
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