SgeI

SgeI is a restriction endonuclease that can cleave DNA targets containing 5-methylcytosine on either single-stranded or double-stranded DNA. It recognizes the m5CNNG(9/13) site and exhibits optimal digestion efficiency in its unique reaction buffer at 37℃. To ensure consistent performance, the enzyme storage buffer contains BSA, which enhances enzyme stability and binds to any potential contaminants in the DNA preparation.Enzyme activity assayPrecautions：The restriction enzyme should be kept on ice during use, and stored at -20℃ immediately after use.The SgeI requires at least 2 recognition sites in the substrate DNA for efficient cleavage.Complete digestion of methylated DNA by SgeI depends on the number of recognition sites in the DNA. Additionally, the products generated from the specific cleavage can promote non-specific cleavage by SgeI. Therefore, it is recommended to optimize the amount of SgeI for the digestion reaction.For ultrapure water without nuclease, we recommend the Pure™ Ultrapure Water (DNase/RNase-Free, Sterile) (, ST876).If the expected enzyme digestion site cannot be cut, please check for potential methylation interference.Isoschizomers may have different sensitivities to different methylations. When encountering potential methylation interference issues, a different isochimzomer can be attempted.This product is for R&D only. Not for drug, household, or other uses.For your safety and health, please wear a lab coat and disposable gloves during the operationInstructions for Use：1. Set up the single enzyme digestion reaction on ice as follows:ReagentPlasmid DNAUltrapure Waterto 20µl10X SgeⅠ Buffer2µlSubstrate DNAxµl (up to 0.5- 2µg)SgeI0.2-1µlTotal volume20µlNote: The reaction volume can be scaled up or down proportionally.a. Mix Gently by pipetting the reaction mixture up and down, or by flicking the reaction tube. Centrifuge briefly to collect the liquid at the bottom of the tube.b. Incubate at 37℃ for 1 hour preferably in a water bath, as the temperature is usually more stable. It is not recommended to exceed 1 hour for the incubation.c. (Optional) Terminate the reaction by heating at 80℃ for 20 minutes to inactivate the enzyme.2. When performing double or multiple enzyme digestion, the following principles can be referred for reaction setup based on the single enzyme digestion reaction provided above.a. Add 1μl of each restriction enzyme in the reaction. The reaction volume can be scaled up proportionally if needed.b. The total volume of restriction enzymes added in the reaction should not exceed 1/10 of the total reaction volume.c. If the optimal working temperature of different restriction enzymes used is different, start digestion with the enzyme that has a lower optimal temperature, then add other enzymes to perform the digestion at higher temperatures.