Restriction Enzymes BstEII / Eco91I

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 inclue: 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 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, 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 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

Protocol tips
The restriction endonucleases BamHI, BstPI, DraI, EcoRI, Sau3AI, and SmaI (Takara Shuzo Co.); DpnI, HincII, and HindIII (New England BioLabs); and EcoRV (Nippon Gene) were used. Phage T4 DNA ligase, the Klenow fragment of E. coli DNA polymerase I, alkaline phosphatase (E. coli C75) (Takara Shuzo Co.), and Si nuclease (Sankyo Co.) were also used. The reaction conditions for these enzymes were as recommended by the suppliers.
Eco91I (BstEII) (10 U/µL)

Thermo Fisher Scientific

Protocol tips
PCR product was digested by Eco91I restriction enzyme (Thermo Scientific, Dreieich, Germany) at 37 °C for 30 min according to the procedure provided by the manufacturer. Digested products were separated by electrophoresis on a 2% agarose gel in 0.5× of TBE buffer which was stained with ethidium bromide and visualized with a UV transilluminator [15, 16].
FastDigest Eco91I

Thermo Fisher Scientific

Protocol tips
Amplified fragments were digested with HaeIII and Eco91I (BstEII) restriction enzymes (FastDigest), under conditions recommended by the manufacturer (ThermoScientific), separated by electrophoresis in 4% agarose gels, and visualized by staining with ethidium bromide (0.5 μg/mL) and exposure to UV light (λ = 320 nm).
BstEII-HF®

New England BioLabs

Protocol tips
Reactions were stopped during linear amplification, and the amplified library was then purified with the QIAquick PCR purification kit (Qiagen, Hilden, Germany), digested with BstEII-HF and EagI-HF (New England Biolab, Ipswich, MA), and purified again.
BstEII NEB#R0162

New England BioLabs

Protocol tips
To release the insert, the TOPO clones were then digested by BstEII and NcoI (NEB, Ipswich, MA). After gel purification, these inserts were ligated using T4 ligase (NEB, Ipswich, MA) into TN6-GFP previously cut with BstEII and NcoI.
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