Site Directed Mutagenesis (SDM) Human Point mutation THP-1

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.

As autophagy is a multi-step process which includes not just the formation of autophagosomes, but most importantly, flux through the entire system, including the degradation upon fusion with lysosomes, which makes it quite challenging for detection. There are several methods for detection in mammalian cells, including immunoblotting analysis of LC3 and p62 and detection of autophagosome formation/maturation by fluorescence microscopy, Currently, there is no single “gold standard” for determining the autophagic activity that is applicable in every experimental context, hence it is recommended to go for the combined use of multiple methods to accurately assess the autophagic activity in any given biological setting.

When extracting nucleic acids from cell cultures, thorough homogenization of cells via vortexing in lysis buffer is very necessary. Choose the best RNA isolation method keeping in mind the downstream applications, generally, column-based isolations result in clean and concentrated RNA samples. Downstream applications like sequencing and cDNA synthesis require high-quality RNA, always treat the samples with DNases and check their integrity by running a gel.

Get tips on using MagCellect Human B Cell Isolation Kit to perform Cell Isolation B cell

Get tips on using B Cell Isolation Kit II, human to perform Cell Isolation B cell

Get tips on using MACSprep™ PBMC Isolation Kit, human to perform Cell Isolation PBMC Isolation

Get tips on using Human/Mouse Active Caspase-3 Antibody to perform Western blotting Caspase-3

Get tips on using ScriptSeq Complete Kit (Human/Mouse/Rat) to perform RNA sequencing Mouse - J774

Get tips on using Quantifiler™ Human DNA Quantification Kit to perform DNA quantification Brain tissue

Get tips on using PE Mouse Anti-Human CD61 Clone VI-PL2 to perform Flow cytometry Anti-bodies Human - CD61