RNA isolation / purification Cells primary

Get tips on using DCFDA - Cellular Reactive Oxygen Species Detection Assay Kit to perform ROS assay cell type - human primary corneal epithelial cells

Get tips on using Mammary Epithelial Cell Growth Medium to perform 3D Cell Culture Media Human primary breast ephitelial cells-organoids

Get tips on using Mammary Epithelial Cell Growth Medium to perform 3D Cell Culture Media Human primary breast ephitelial cells-Mammospheres

Get tips on using Gibco DMEM/F-12, HEPES to perform 3D Cell Culture Media Human primary breast ephitelial cells-organoids

Get tips on using MammoCult™ Human Medium Kit to perform 3D Cell Culture Media Human primary breast ephitelial cells-Mammospheres

Get tips on using Gibco DMEM/F-12, HEPES to perform 3D Cell Culture Media Mouse primary mammary ephitelial cells- organoids

Get tips on using Dual-Luciferase® Reporter Assay System to perform Reporter gene assay luciferase - primary human endometrial stromal cells

Get tips on using FreeStyle™ 293-F Cells to perform Protein expression and purification Mammalian cells - HEK 293 EGFR

The RNA interference (RNAi) is used to inhibit gene expression or translation, by neutralizing targeted mRNA molecules. Two types of RNA molecules such as microRNA (miRNA) and small interfering RNA (siRNA) play a central role in RNAi. Few points have to considered to increase the transfection efficiency of siRNA. Always use healthy, actively dividing cells to maximize transfection efficiency. The confluency of cells should be between 50-70%. Always use the most appropriate siRNA concentration to avoid off-target effects and unwanted toxic side effects. Positive and negative controls should be used for each and every experiment to determine transfection efficiency.

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.