PureLink™ HiPure Plasmid DNA Purification Midiprep Kit From Invitrogen

A multitude of kits are now available for the researcher wishing to purify large amounts of plasmid DNA. Invitrogen has added another option, with its PureLink™ HiPure Plasmid DNA Purification Midiprep Kit. The PureLink™ HiPure Plasmid DNA Purification Midiprep Kit is available in sizes of 25 or 50 preps. The midiprep kit is designed for bacterial cultures starting at 15-25 ml for high copy number plasmids or up to 100 ml for low copy number plasmids. According to the manufacturer, the kit can isolate 100-300 µg of DNA in around 2 hours. The kit may be used to purify BAC, cosmid, bacmid and ssM13 DNA, using slightly modified protocols supplied in the manual.

The protocol of the Invitrogen kit will be familiar to any researcher who has used a plasmid DNA purification kit before. A culture of bacterial cells is grown overnight, then harvested by centrifugation. The pellet is resuspended in a buffer containing RNAse A (R3), then the mixture is incubated for 5 minutes with a lysis buffer (L7). A precipitation buffer (N3) is added and after mixing carefully, the lysate is clarified by centrifugation for 10 minutes. The DNA purification process is based around an anion exchange resin prepacked into a column, which is equilibrated while the bacteria are harvested (Buffer EQ1). The supernatant from the clarified lysate is loaded onto this column, which is drained by gravity flow. During this step, the resin binds the plasmid DNA. The column is then washed several times to remove impurities and finally the DNA is eluted using buffer E4. The DNA is precipitated from the final solution using isopropanol via a 30 minute centrifugation step and an alcohol wash. After drying at room temperature, the DNA pellet is finally resuspended in 200µl TE buffer.

There are a few design issues with this kit that need to be highlighted. The column processing steps (equilibration, loading supernatant, washing, elution) are annoyingly slow and the comparatively small diameter and deep resin reservoir of the columns probably contribute to this. The columns do seem to be very sensitive to clogging when the supernatant is added, particularly when larger cultures are used. (Invitrogen now supply Plasmid Purification kits with filters which address this problem.) The columns don’t sit well in standard tubes so the Purification Rack (purchased separately) could be useful for those doing large numbers of preps. The isopropanol precipitation step is also lengthy and the Precipitators which can be used to speed up this final step must be purchased separately. Other midiprep kits in the same price range include the Precipitators as standard. Finally, if doing the isopropanol precipitation, the final resuspension often contains a white precipitate. This can be removed by centrifugation, however, why it occurs is not addressed.

In all fairness, the quality of the DNA obtained using these kits is quite good. I have purified plasmids up to 10 kb with no problems (isopropanol precipitation only) and yields are always excellent. I have subsequently used the DNA in restriction digests, sequencing and transfections with good results, and have not had issues with sterility, stability or endotoxins.