rna-isolation-purification-cells-immortalized-ags

Get tips on using ApoAlert™ DNA Fragmentation Assay Kit to perform TUNEL assay cell type - Islets of langerhans (Beta cells)

Get tips on using ApoAlert™ DNA Fragmentation Assay Kit to perform TUNEL assay cell type - HeLa cells human cervical cancer

Get tips on using Cell Counting Kit-8 to perform Cell cytotoxicity / Proliferation assay cell type - HT22 mouse hippocampal cells

Get tips on using Low Input Quick Amp Labeling Kits to perform Microarray RNA amplification & Labeling - Fish fundulus heteroclitus Cyanine-3 / Cyanine-5

Get tips on using Amino Allyl MessageAmp™ II aRNA Amplification Kit to perform Microarray RNA amplification & Labeling - Human brain tissue Cyanine 3

Get tips on using Thiazolyl Blue Tetrazolium Bromide to perform Cell cytotoxicity / Proliferation assay cell type - HT22 mouse hippocampal cells

Get tips on using Live/Dead Cell Double Staining Kit to perform Live / Dead assay mammalian cells - L29 mouse fibroblast

Get tips on using Live-Dead cell staining kit (Enzo) to perform Live / Dead assay mammalian cells - human fibroblast tissue

Get tips on using Dual-Luciferase® Reporter Assay System to perform Reporter gene assay luciferase - primary human endometrial stromal cells

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.