Site Directed Mutagenesis (SDM) Hamster Point mutation CHO

Get tips on using Lipofectamine® 2000 Transfection Reagent to perform DNA transfection Mammalian cells - Immortalized cell lines CHO

Get tips on using Luminescent β-galactosidase Detection Kit II to perform Reporter gene assay β-galactosidase substrates - CHO

Get tips on using ExpiCHO™ Expression System Kit to perform Protein expression and purification Mammalian cells - CHO-K1 SUMO protein

Get tips on using CytoTox 96® Non-Radioactive Cytotoxicity Assay to perform Live / Dead assay mammalian cells - CHO-K1

Get tips on using mirVana™ miRNA Isolation Kit, with phenol to perform RNA isolation / purification Cells - immortalized CHO-K1

Get tips on using pcDNA™3.1 (+) Mammalian Expression Vector to perform Protein expression and purification Mammalian cells - CHO-K1 sRAGE

Get tips on using Senescence β-Galactosidase Staining Kit - Cell Signaling to perform Reporter gene assay β-galactosidase substrates - CHO-K1

Get tips on using LIVE/DEAD™ Viability/Cytotoxicity Kit, for mammalian cells to perform Live / Dead assay mammalian cells - CHO-K1

Get tips on using β-Galactosidase Enzyme Assay System with Reporter Lysis Buffer to perform Reporter gene assay β-galactosidase substrates - CHO-K1

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.