HiSpeed Plasmid Maxi Kit From Qiagen

One of the exceptional features of the HiSpeed Plasmid Maxi Kit from Qiagen is that it involves no centrifugation step (except to pellet the bacterial cells). The QIAfilter Cartridges enable rapid and efficient clearing of bacterial lysates without repeated centrifugation. The QIAprecipitator facilitates DNA precipitation; less than 5 min is required for isopropanol precipitation and ethanol washes, and there is no risk of DNA pellet loss during precipitation. In addition to quick plasmid purification, the elimination of repeated centrifugation increases the percentage of super coiled plasmid DNA. The purification protocol yields approximately 700 µg of high-grade purified supercoiled plasmid DNA from 250 ml of bacterial culture (for a high copy number plasmid grown in E. coli DH5 for 16-18 hr). The HiSpeed Kit is suitable for isolation of both high- and low-copy plasmids. However, in the case of low-copy plasmids, since the quantity of plasmid is limited, yields will be lower. The other great feature of the kit is the elimination of various toxic reagents, such as phenol and chloroform. The entire purification process takes less than 1 hour.

The protocol begins with the pelleting of bacterial culture followed by resuspension of the pellet in Buffer P1. The lysis and neutralization steps can be followed by color coded buffers where the lysis buffer makes the solution blue; the color then returns to white upon addition of neutralization buffer. It is important not to exceed incubation with the lysis buffer for more than 5 min as extended incubation can result in irreversible denaturation of plasmid DNA.

The QIAfilter columns are then used to clear the cellular debris and the filtrate is directly passed through the anion-exchange column by gravity flow. The column is then washed and the bound DNA is eluted with high salt buffer and precipitated with isopropanol. The DNA-containing isopropanol solution is then loaded onto the QIAprecipitator filter-shaped column, avoiding the lengthy centrifugation step normally required for isopropanol precipitation. The QIAprecipitator then goes through an ethanol wash, which again involves no centrifugation and thus no possibility of loss of DNA pellet. The bound DNA is eluted with 500 l of water or Tris buffer.

In my experience, the HiSpeed-purified plasmid DNA is the best for transfection of recombinant adenoviral constructs for generating recombinant adenoviruses. The kit is very reliable, fast and produces good quality plasmid DNA.