MagNA Pure LC DNA Isolation Kit 1 From Roche Applied Science

The MagNa Pure LC DNA Isolation System is designed to isolate DNA with a high purity from mammalian whole blood, cells or cultured cells. The LC DNA Isolation Kit 1 must be used in conjunction with the Roche MagNA Pure Instrument; the principle of DNA isolation is based on magnetic bead technology. Briefly, the samples are first lysed by incubation with a buffer containing chaotropic salts and Proteinase K. Magnetic Glass Particles (MGP) are then added and the DNA binds to their surfaces. Unbound substances are removed by several washing steps and the purified DNA is eluted. DNA may be isolated from 20-200 ìl mammalian whole blood or 1 x 10³ - 1 x 10⁶ mammalian blood cells (white blood cells [WBCs] or peripheral blood mononuclear cells [PBMCs], or cultured cells) and the resultant DNA is suitable for use in downstream RT-PCR reactions.

The main advantage of the system is that it is fully automated, thus requiring minimal hands-on time. The sample material must be placed into the wells of the sample cartridge and the sample names are entered into the on-screen system. The Stage Setup program then tells the user exactly which plastics and solutions need to be loaded into the MagNA Pure instrument and all reagents are color coded to aid with setup. Included with the LC DNA Isolation Kit I are the MGP’s, Wash Buffer I, Wash Buffer II, Elution Buffer, Lysis/Binding Buffer and Proteinase K. The user must supply the plastics and tips required. Once all reagents and materials are in place and the sample names have been entered, the user starts the automated program and no further action is required. The system also gives an estimated time of completion.

The principle steps of the isolation procedure first involve loading the sample into the Sample Cartridge. The MagNA Pure instrument automated program then adds Lysis/Binding Buffer to each sample, resulting in complete cell lysis and releasing all nucleic acids. The nucleases are denatured and addition of Proteinase K acts to digest proteins. Due to the chaotropic salt conditions, isopropanol, and the high ionic strength of the Lysis/Binding Buffer, the DNA binds to the silica surface of the added MGPs and MGPs with bound DNA are magnetically separated from the lysed sample. The MGPs with bound DNA are then washed a number of times with Wash Buffer to remove unbound substances (such as proteins, cell membranes, PCR inhibitors) and to reduce the chaotropic salt concentration. Purified DNA is then eluted at 70°C into the Elution Cartridge, whereas the MGPs are retained in the reaction tip and discarded.

I use the MagNa Pure LC DNA Isolation Kit 1 to isolate DNA from cultured cell-line cells in suspension. I frequently obtain high quality DNA with optimal 260/280 ratios. I have obtained up to 300 ng/ul using 10⁶ cultured cells and I have encountered no problems when using the resultant DNA for qRT-PCR.