Protein Expression Prokaryotic cells L. citreum

Get tips on using JetPrime to perform DNA transfection Mammalian cells - Primary cells Human lung fibroblasts (HLF)

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.

Get tips on using Lipofectamine® 2000 Transfection Reagent to perform DNA transfection Mammalian cells - Primary cells Cardiomyocytes

Get tips on using X-tremeGENE™ HP DNA Transfection Reagent to perform DNA transfection Mammalian cells - Primary cells Human aortic smooth muscle cells (HOSMC)

Get tips on using TRIzol Reagent to perform RNA isolation / purification Cells - primary human lung microvascular endothelial cells

Get tips on using miRNeasy Mini kit to perform RNA isolation / purification Cells - primary rat lymph node cells

Get tips on using QIAamp DNA Blood Mini Kit to perform DNA isolation / purification Cells - Primary cells Lymphocytes

Get tips on using PolyFect Transfection Reagent to perform DNA transfection Mammalian cells - Immortalized cell lines Chang Liver cells

Get tips on using Genomic DNA isolation to perform DNA isolation / purification Cells - Immortalized cell lines Leiomyoma & myometrial cells

Get tips on using Lipofectamine® 2000 Transfection Reagent to perform DNA transfection Mammalian cells - Primary cells Rat cardiomyocytes