RNA sequencing Mouse

Get tips on using Lipofectamine® 2000 Transfection Reagent to perform siRNA / miRNA gene silencing Human - Primary Endometrial Stromal Cells IGFBP1 (Insuline-like growth factor binding protein-1) Lipid

Get tips on using CD171 (L1CAM) Antibody, anti-human, PE-Vio® 770, REAfinity™ to perform Flow cytometry Anti-bodies Human - CD171/L1CAM

Get tips on using LSI VetMAX™ Bluetongue Virus Real-Time PCR Kit, BTV1 typing - IAH to perform Cell Culture Contamination Detection Kit Virus

Get tips on using Image-IT™ LIVE Green Reactive Oxygen Species Detection Kit, for microscopy to perform ROS assay cell type - MCF-7

Get tips on using DCFDA - Cellular Reactive Oxygen Species Detection Assay Kit to perform ROS assay cell type - PLHC-1, SK-HEP-1, Hep3b, HepG2 human hepatocellular carcinoma

Get tips on using ViralSEQ™ Quantitative Sf-Rhabdovirus Kit with PrepSEQ™ Nucleic Acid Sample Prep Kit to perform Cell Culture Contamination Detection Kit Virus

Get tips on using SNP Type™ 96.96 Genotyping Reagent Kit with Control Line Fluid—10 IFCs to perform Cell line authentication Human prostatic cancer cell line DU145

Get tips on using SNP Type™ 96.96 Genotyping Reagent Kit with Control Line Fluid—10 IFCs to perform Cell line authentication Human prostatic cancer cell line PC3

ELISA is the most commonly used method of detecting and quantifying the concentration of an antigen in an unknown sample. During the experiment, If you get a weak signal, then make sure reagents are at room temperature before starting the assay. Try increasing incubation times to ensure maximal antibody binding and amplify the signal. Secondly, if you get values above 0 in the negative control indicates a high background signal. Try to consider reducing your antibody concentration and prevent non-specific binding of antibodies by using affinity-purified antibody and suitable blocking buffers. To avoid high well to well variation, do not stack plates during incubation, no bubbles in the plate and wash wells thoroughly to avoid variation.

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.