Site Directed Mutagenesis (SDM) Human Point mutation HUVEC

Get tips on using Direct-zol RNA Kits to perform RNA isolation / purification Cells - immortalized PC-3

Get tips on using Direct-zol RNA Kits to perform RNA isolation / purification Bacteria - Gram positive Streptococcus pyogenes

Get tips on using EZ DNA Methylation-Direct Kit to perform PCR Methylation specific PCR - Mammalian DNA

Get tips on using MagAttract Direct mRNA M48 Kit (192) to perform mRNA / Ribonucleoprotein isolation / purification mRNA

Get tips on using EZ DNA Methylation-Direct Kit to perform DNA methylation profiling Gene specific profiling - A54 RASSF1A

Get tips on using Direct-zol™ RNA MiniPrep Plus to perform RNA isolation / purification Bacteria - Gram negative Borrelia burgdorferi

Get tips on using EZ DNA Methylation-Direct Kit to perform DNA methylation profiling Gene specific profiling - A2780 miR-30a-5p

Get tips on using EZ DNA Methylation-Direct Kit to perform DNA methylation profiling Gene specific profiling - A2780 miR-30c-5p

Get tips on using Direct-zol™ RNA MiniPrep Plus to perform RNA isolation / purification Bacteria - Gram negative Escherichia coli

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.