Site Directed Mutagenesis (SDM) Mouse Neuroblastoma 2a

Gene silencing through the use of small interfering RNA (siRNA) has become a primary tool for identifying disease-causing genes. There are several aspects for preparing and delivering effective siRNA to knockdown a target gene. The length of siRNA should be 21–23nt long with G/C content 30–50%. If a validated siRNA sequence for your target gene is not available, use siRNA generated against the entire target gene ORF. Always work with two or three different siRNA constructs to get reliable results. If you are not sure how much siRNA to use for a given experiment, start with a transfection concentration of 10-50 nM and use siRNA-specific transfection reagent to ensure efficient siRNA delivery in a wide range of cells.

Get tips on using ChIP Kit Magnetic - One Step (ab156907) to perform ChIP Human - SMMC-7721

Get tips on using SimpleChIP® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003 to perform ChIP Human - SMMC-7721

Western blotting is a widely used technique to size separate proteins from a pool of cell or tissue lysates. The technique has 4 major steps: a) gel electrophoresis, b) blocking and treatment with antigen specific antibody, c) treatment with secondary antibody and finally d) detection and visualization. Though western blotting is a widely used technique, detection of specific proteins depends on several factors, the major ones are antibody concentration, incubation time and washing steps. Key points for obtaining clean blots are: always prepare fresh buffer solutions and optimize antibody concentration. Given the advent of high-throughput protein analysis and a push to limit the use of lab consumables, onestep antibodies are developed which recognise protein of interest and also contain a detection label.

Western blotting is a widely used technique to size separate proteins from a pool of cell or tissue lysates. The technique has 4 major steps: a) gel electrophoresis, b) blocking and treatment with antigen specific antibody, c) treatment with secondary antibody and finally d) detection and visualization. Though western blotting is a widely used technique, detection of specific proteins depends on several factors, the major ones are antibody concentration, incubation time and washing steps. Key points for obtaining clean blots are: always prepare fresh buffer solutions and optimize antibody concentration. Given the advent of high-throughput protein analysis and a push to limit the use of lab consumables, onestep antibodies are developed which recognise protein of interest and also contain a detection label.

Western blotting is a widely used technique to size separate proteins from a pool of cell or tissue lysates. The technique has 4 major steps: a) gel electrophoresis, b) blocking and treatment with antigen specific antibody, c) treatment with secondary antibody and finally d) detection and visualization. Though western blotting is a widely used technique, detection of specific proteins depends on several factors, the major ones are antibody concentration, incubation time and washing steps. Key points for obtaining clean blots are: always prepare fresh buffer solutions and optimize antibody concentration. Given the advent of high-throughput protein analysis and a push to limit the use of lab consumables, onestep antibodies are developed which recognise protein of interest and also contain a detection label.

Western blotting is a widely used technique to size separate proteins from a pool of cell or tissue lysates. The technique has 4 major steps: a) gel electrophoresis, b) blocking and treatment with antigen specific antibody, c) treatment with secondary antibody and finally d) detection and visualization. Though western blotting is a widely used technique, detection of specific proteins depends on several factors, the major ones are antibody concentration, incubation time and washing steps. Key points for obtaining clean blots are: always prepare fresh buffer solutions and optimize antibody concentration. Given the advent of high-throughput protein analysis and a push to limit the use of lab consumables, onestep antibodies are developed which recognise protein of interest and also contain a detection label.

Isolating RNA from tissues and paraffin-embedded tissue samples can be challenging due to cross-linking of biomolecules and fragmented nucleic acids. The best solution is to slice the tissues into smaller pieces and make a homogenate solution (using tissue homogenizer or grinding liquid nitrogen frozen samples) in presence of RNAse inhibitors. The homogenization process should be carried out on dry ice to maintain the integrity of RNA.

Reporter gene assays are designed to test the regulation of the expression of a gene of interest. This is usually done by linking the promoter of the gene of interest with a gene such as a firefly luciferase, which can be easily detected by addition of luciferin that leads to an enzymatic reaction to produce luminescence. The enzymatic reaction can be correlated to the expression of the gene of interest. Another luciferase gene that can be used is Renilla luciferase. For an appropriate luciferase assay: 1. the reporter should express uniformly in all cells, 2. specifically respond to effectors that the assay intends to monitor, 3. have low intrinsic stability to quickly reflect transcriptional dynamics. It is important to have an equal number of cells plated in each testing condition to avoid any incorrect readouts. Reporter assays could be single or dual reporter assays. The reporter could be both luciferases. Most dual-luciferase assays involve adding two reagents to each sample and measuring luminescence following each addition. Adding the first reagent activates the first luciferase reporter reaction; adding the second reagent extinguishes first luciferase reporter activity and initiates the second luciferase reaction. Dual-luciferase assays have some advantages, including 1. reduces variability, 2. reduces background, 3. normalizes differences in transfection efficiencies between samples.

DNA-protein interactions are studied by using ChIP. The basic steps in this technique are crosslinking, sonication, immunoprecipitation, and analysis of the immunoprecipitated DNA. During ChIP, if chromatin is under-fragmented or fragments are too large which can lead to the increased background and lower resolution. Shorter cross-linking times (5-10 min) and/or lower formaldehyde concentrations (<1%) may improve shearing efficiency. If Chromatin is over-fragmented, then optimize shearing conditions for each cell type to improve ChIP efficiency. Over-sonication of chromatin may disrupt chromatin integrity and denature antibody epitopes. If you do not see any product or very little product in the input PCR reactions, add 5–10 μg chromatin per IP.