RNA

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The estimation of DNA methylation level heavily depends on the complete conversion of non-methylated DNA cytosines. It is crucial to ensure complete conversion of non-methylated cytosines in DNA. Therefore, it is important to incorporate controls for bisulfite reactions, as well as to pay attention to the appearance of cytosines in non-CpG sites after sequencing, which is an indicator of incomplete conversion.

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 ELISA Kit for C Reactive Protein (CRP) to perform ELISA Rat - C-Reactive Protein/CRP

Get tips on using CelLytic™ MT Cell Lysis Reagent to perform Protein isolation Mammalian cells - Rat_Mesenteric fat

Get tips on using CelLytic™ MT Cell Lysis Reagent to perform Protein isolation Tissue - Rat skin tissue

Get tips on using CelLytic™ MT Cell Lysis Reagent to perform Protein isolation Tissue - Rat prostate tissue

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

Get tips on using FuGENE® 6 Transfection Reagent to perform DNA transfection Mammalian cells - Primary cells Rat cardiomyocytes

Get tips on using FuGENE® HD Transfection Reagent to perform DNA transfection Mammalian cells - Primary cells Rat microglia