Immunohistochemistry Collagen VII [II-32] Mouse

Get tips on using CellGenix® GMP SCGM Stem Cell Growth Medium to perform Mammalian cell culture media NK-92

Get tips on using TRI Reagent® to perform Protein isolation Mammalian cells - Mouse_Brown fat

Get tips on using Mem-PER™ Plus Membrane Protein Extraction Kit to perform Protein isolation Mammalian cells - Mouse_Brown fat

Get tips on using FastDigest BpiI (IIs class) to perform Restriction Enzymes BbsI

Get tips on using FastDigest Eco31I (IIs class) to perform Restriction Enzymes BsaI / Eco31I

Get tips on using SuperScript™ III Reverse Transcriptase to perform AAA for reviews

Get tips on using MagNA Pure LC RNA Isolation Kit III to perform RNA isolation / purification Tissue - Human ileum

Get tips on using In Vitro ROS/RNS Assay to perform ROS assay cell type - human umbelical vein endothelial cells (HUVEC)

Get tips on using OxiSelect™ In Vitro ROS/RNS Assay Kit (Green Fluorescence) to perform ROS assay cell type - human umbelical vein endothelial cells (HUVEC)

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.