siRNA / miRNA gene silencing Human ACC-MESO1

Get tips on using Nucleic Acid Purification to perform Plasmid Isolation DH10Bac (Bacmid)

Get tips on using Trichloroacetic acid to perform Protein isolation Mammalian cells - THP-1

Get tips on using AcP(−)p6.9hSEAP to perform Protein Expression Eukaryotic cells - S. frugiperda hSEAP

Get tips on using AcP(−)p6.9hEPO to perform Protein Expression Eukaryotic cells - S. frugiperda hEPO

Get tips on using Anti-Choline Acetyltransferase Antibody to perform Immunohistochemistry Mouse - ChAT

Get tips on using NOTCH1/activated Notch1 Antibody to perform Immunohistochemistry Mouse - Notch1

Get tips on using Anti-Choline Acetyltransferase Antibody AB5042 to perform Immunohistochemistry Mouse - ChAT

Get tips on using Anti-activated Notch1 antibody (ab8925) to perform Western blotting Notch1

Get tips on using Mouse Adiponectin/Acrp30 DuoSet ELISA to perform ELISA Mouse - Adiponectin

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.