siRNA / miRNA gene silencing Human Min-6

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 GeneJET RNA Purification Kit to perform AAA for reviews

Get tips on using GeneRead DNA FFPE Kit to perform DNA isolation / purification Tissue - FFPE samples

Get tips on using GenePrint® 10 System to perform Cell line authentication MCF-7 cell line

Get tips on using VRESelect Medium #63751 to perform Bacterial cell culture media Enterococcus faecium

Get tips on using VRESelect Medium #63751 to perform Bacterial cell culture media Enterococcus faecalis

Get tips on using CM-H2DCFDA (General Oxidative Stress Indicator) to perform ROS assay cell type - PC12

Get tips on using CM-H2DCFDA (General Oxidative Stress Indicator) to perform ROS assay cell type - T47D

Get tips on using GeneJuice® Transfection Reagent to perform DNA transfection Mammalian cells - Immortalized cell lines 3T3-L1

TUNEL assay is the cell death detection method where the biochemical marker of apoptosis is DNA fragmentation. The assay involves the microscopical detection of generated DNA fragments with free 3'-hydroxyl residues. in apoptotic cells using enzyme terminal deoxynucleotidyl transferase (TdT) which adds biotinylated nucleotides at the site of DNA breaks. Major challenges of this method involve proper access of the enzyme which could be hampered by poor permeabilization and/or excessive fixation with cross-linking fixative (common with archival tissue). This issue can be resolved by optimizing the incubation time with Proteinase K or CytoninTM.