ROS assay

Get tips on using HincII (HindII) restriction enzyme to perform Restriction Enzymes HincII / HindII

Get tips on using EcoT14I (StyI) restriction enzyme to perform Restriction Enzymes StyI / EcoT14I / Eco130I

Get tips on using VpaK11BI (AvaII) restriction enzyme to perform Restriction Enzymes AvaII / Eco47I / VpaK11BI

Get tips on using Aor51HI (Eco47III) restriction enzyme to perform Restriction Enzymes AfeI / Eco47III / Aor51HI

Get tips on using BlnI (AvrII) restriction enzyme to perform Restriction Enzymes BlnI / AvrII / XmaJI

Get tips on using Ras (D2C1) Rabbit mAb #8955 to perform Western blotting Ras

Get tips on using BstPI (BstEII, EcoO65I) restriction enzyme to perform Restriction Enzymes BstEII / Eco91I

Get tips on using HapII (HpaII, MspI) restriction enzyme to perform Restriction Enzymes HpaII / MspI

Get tips on using K-Ras Antibody (F234): sc-30 to perform Western blotting Ras

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.