rna-isolation-purification-cells-immortalized-mda-mb-361

Get tips on using pBAD–LCY to perform Protein Expression Prokaryotic cells - E. coli Lycopene

Get tips on using pET28a-HsLARP6 to perform Protein Expression Prokaryotic cells - E. coli LARP6

Get tips on using pET44a-CrtY to perform Protein Expression Prokaryotic cells - E. coli CrtY

Get tips on using pXMJ19-xynA to perform Protein Expression Prokaryotic cells - C. glutamicum XynA

Get tips on using pAU5-amyE to perform Protein Expression Prokaryotic cells - C. glutamicum amyE

Get tips on using pBYR2T-EGFP to perform Protein Expression Prokaryotic cells - A. tumefaciens GFP

Get tips on using pFastBac1-TEV-TRPV1 to perform Protein Expression Eukaryotic cells - HEK293 TRPV1

Get tips on using pCMV-HA2/DKK1 to perform Protein Expression Eukaryotic cells - HEK293 DKK1

Get tips on using pgMAX system-DsRed2 to perform Protein Expression Eukaryotic cells - HEK293 DsRed2

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.