DNA Damage Assay Human bronchial epithelial cells (hBE)

Get tips on using RIPA Lysis and Extraction Buffer to perform Protein isolation Mammalian cells - HEK293T

Get tips on using Gibco™StemPro™ Chondrogenesis Differentiation Kit to perform Stem cell Differentiation media hBMSCs differentiation into chondrogenic 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.

Get tips on using HeV NCORE pET43.1a to perform Protein Expression Prokaryotic cells - E. coli HeV N

Get tips on using DuoSet® ELISA Development Systems to perform ELISA (kit) IL-6, IL-1β, IL-8 and TNF-α - -NA- Human -NA-

Get tips on using QuikChange II XL Site-Directed Mutagenesis Kit, 10 Rxn to perform Site Directed Mutagenesis (SDM) Human - Deletion K562 c-Myb gene

Get tips on using QuikChange II XL Site-Directed Mutagenesis Kit, 10 Rxn to perform Site Directed Mutagenesis (SDM) Human - Point mutation U-87MG MEF

Get tips on using QuikChange II XL Site-Directed Mutagenesis Kit, 10 Rxn to perform Site Directed Mutagenesis (SDM) Human - Point mutation SH-SY5Y FGF1

Get tips on using CD49f (Integrin alpha 6) Monoclonal Antibody (eBioGoH3 (GoH3)), eFluor 450, eBioscience™ to perform Flow cytometry Anti-bodies Human - CD49f/ITGA6

Site-directed mutagenesis (SDM) can be challenging, particularly during detection/confirmation of (SDM) in colonies by sequencing or PCR techniques. This common issue in SDM is heavily relying on designing of mutagenic primer pairs. The best solution is to design the mutagenic primers that have extended 3'-ends/3'-overhang. This would provide the annealing region between the mutagenic primer pair is essentially shorter. and hence ensure a lower annealing temperature for the primer pair along with a higher chance of annealing to the template.