siRNA / miRNA gene silencing Human HUVEC IL-8

Cells are sourced from various tissues to grow them in in-vitro conditions. Therefore, cell specific nutrients are important for their survival, maintenance and growth. Determining the appropriate cell culture media is a challenge if you are growing a cell line or a microorganism for the first time. Established cell lines, primary cells, stem cells, bacteria and Yeast all require varied nutrients from basic to complex. Based on the cell type, one can easy find what media and nutrients your peers have used before you try to reinvent the wheel.

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.

A gross majority of classical apoptotic attributes can be quantitatively examined by flow cytometry, the preferred platform for rapid assessment of multiple cellular attributes at a single-cell level. However, sample preparation for such flow cytometry-based techniques could be challenging. Cell harvesting by trypsinization, mechanical or enzymatic cell disaggregation from tissues, extensive centrifugation steps, may all lead to preferential loss of apoptotic cells. To overcome this strictly follow manufacturers instruction of the detection kit.

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