siRNA / miRNA gene silencing Human Primary Endometrial Stromal Cells hsa-miR-542-3p

Plasmid isolation is an important technique in molecular biology or any kind of genetic editing. It involves amplifying plasmids overnight by transforming them into competent bacterial cells. The desired colonies of these bacteria can then be grown in shaker cultures, at appropriate shaking speed, oxygen availability and temperature. These liquid cultures can then be ultracentrifuged to pellet the bacteria, which are then used for plasmid isolation. The bacteria are first resuspended in a buffer, then lysed, neutralized, purified in a column, eluted, precipitated with ethanol and then resuspended. During plasmid isolation, it is important to lyse cells quickly because lysing bacteria for too long may lead to irreversible denaturing of the plasmid. Usually, alkaline lysis is used for isolation because it is a mild treatment. It isolates plasmid DNA and other cell components such as proteins by breaking cells apart with an alkaline solution. Precipitation removes the proteins, and the plasmid DNA recovers with alcohol precipitation. Resuspension and lysis buffers should be mixed thoroughly in order to prevent the DNA from breaking into smaller fragments. This is because broken gDNA can reanneal and remain in the solution, without binding to the column.

Get tips on using RNeasy Micro Kit to perform RNA isolation / purification Tissue - Human Tongue

Get tips on using RNeasy Micro Kit to perform RNA isolation / purification Tissue - Human Eye

Get tips on using RNeasy Micro Kit to perform RNA isolation / purification Tissue - Human Bone

Get tips on using RIP-Assay Kit for microRNA to perform ChIP Human - MCF-7

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.

Get tips on using KAPA Stranded mRNA-Seq Kit to perform RNA sequencing Human - SKBR-3

Get tips on using KAPA Stranded mRNA-Seq Kit to perform RNA sequencing Human - MCF-7

Get tips on using β-Galactosidase Enzyme Assay System with Reporter Lysis Buffer to perform Reporter gene assay β-galactosidase substrates - SK-Hep-1

Get tips on using pLV hUbC-dCas9-T2A-GFP to perform CRISPR Human - Repression HS2