PureLink™ HiPure Plasmid Filter Maxiprep Kit From Invitrogen

The PureLink™ HiPure Plasmid Maxiprep Kit from Invitrogen is designed to isolate plasmid DNA of the highest purity from 25 to 500 mls of bacterial culture. The amount of culture is dependent on plasmid copy number. I have found that 150 mls of culture of a high copy plasmid will yield between 850 micrograms and 1 milligram of total DNA with an A260/A280 ratio of ~1.9.

The kit works based upon anion-exchange technology in which plasmid DNA is bound to resin in low/medium salt followed by elution in high salt (after several washes) and precipitation in isopropanol to remove the salt. The DNA can then be resuspended in a functional buffer such as Tris-EDTA (TE). The total prep time from start to finish is approximately 2 hours for experienced users. I would allow an extra 30-45 minutes for first time users.

The kit includes the HiPure Filter Columns, which combine filtration for clarification of the bacterial lysate with anion-exchange resin purification of plasmid DNA, in a single unit. Upon cell lysis and precipitation of proteins and genomic DNA, the entire mixture is poured into the column. The filtration portion of the column prevents the precipitated material from passing through while allowing the supernatant to pass through into the anion-exchange portion of the column. After the supernatant has passed through, the filtration portion is removed and discarded. Standard washes are then performed followed by elution of DNA from the anion-exchange resin. The biggest advantage to the filter columns is the time saved because you do not need to centrifuge to pellet the precipitated protein and genomic DNA before applying the supernatant to the column as with traditional preps.

According to Invitrogen, the resulting plasmid DNA is of a quality equivalent to that obtained by purification with two passes through cesium chloride gradients - the most rigorous method for DNA purification. Although I have not directly performed this comparison, I do find the plasmid DNA perfectly suitable for transfection of mammalian tissue culture cells and have never had issues with endotoxins. Additionally, the elimination of phenol, chloroform, ethidium bromide, and cesium chloride from the protocol minimizes exposure to, and disposal of hazardous materials.