rna-isolation-purification-cells-immortalized-brl-3a

Get tips on using EZ DNA Methylation kit to perform DNA methylation profiling Whole genome profiling - mouse hematopoietic stem cells

Get tips on using pET-Sac-Aβ(M1–42) to perform Protein Expression Prokaryotic cells - E. coli Aβ(M1–42)

Get tips on using pET-21b(+)/Pro j 1 to perform Protein Expression Prokaryotic cells - E. coli Pro J 1

Get tips on using ON-TARGETplus Mouse Jun siRNA to perform siRNA / miRNA gene silencing Mouse - Embryonic stem cells Jun

Get tips on using ON-TARGETplus Mouse Gpam siRNA to perform siRNA / miRNA gene silencing Mouse - Embryonic stem cells Gpat1

Get tips on using pSpCas9(BB)-2A-Puro (PX459) to perform CRISPR Mouse - Deletion ES (embryonic stem) cells Etv2 promoter

Get tips on using His-Strep pQE-TriSystem Vector Set to perform Protein Expression Prokaryotic cells - E. coli Integrin αV

Get tips on using pcDNA™3.1D/V5-His TOPO®-hsEH to perform Protein Expression Eukaryotic cells - HEK293 hsEH

Get tips on using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003 to perform ChIP Mouse - 3T3-L1 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 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.