Restriction Enzymes FokI

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 FokI

Thermo Fisher Scientific

Protocol tips
After the production of PCR products, FokI and ApaI restriction enzymes (Thermo Fisher Scientific, USA) and enzyme buffer were used for digestion of PCR products (RFLP or Restriction fragment length polymorphism). FokI PCR product was digested for 2 hours in 55°C, ApaI PCR product was digested for 2 hours in 37°C, and electrophoresis of digestion products on 2.5% agarose gel was used to determine FokI and ApaI polymorphisms. For FokI site, 265 bp and 169+96 bp represented alleles C and T, respectively (Fig. 1). For ApaI, 740 bp and 530+210 bp represented alleles A and C, respectively (Fig. 2).
Protocol tips
3 jig RNA was converted to double-stranded cDNA with 200 U reverse transcriptase, and digested with 5 U of either of three class IIS restriction enzymes according to the supplier's protocol for 45 min. We used FokI (TaKaRa), BsmAI (NEB) and BsmFI (NEB). After phenol extraction and ethanol precipitation, the product was dissolved in 70 gl of distilled water.
FokI NEB#R0109

New England BioLabs

Protocol tips
After PCR amplification, samples are subjected to restriction enzyme digestion with FokI and BtsCI and desalting using Oasis purification plates, followed by analysis by MALDI-TOF MS.
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