NucleoBond® Xtra Plasmid Midi Kit From Clontech

This article introduces yet another option for the researcher requiring large amounts of purified plasmid DNA – The NucleoBond® Xtra Plasmid Midi Kit. This is an updated version that has been designed for faster processing and increased DNA yields.

This kit enables users to purify 20-250 µg of plasmid DNA, with the maximum capacity of the columns being slightly higher than other available midiprep kits. For high copy plasmids, the manufacturers recommend a maximum culture size of 30 ml and 100 ml for low copy number plasmids. The columns are suitable for plasmids from 3-10 kb, cosmids from 10-50 kb and larger constructs such as bacmid DNA, which can be up to 100 kb in size. The kit is available in 10, 50 and 100 prep sizes and can be stored at room temperature with all components stable for 2 years.

The NucleoBond® Xtra Plasmid Midi Kit purifies DNA using anion-exchange technology. The procedure is similar to other methods of collecting plasmid DNA – First, an overnight culture of cells is pelleted and an alkaline lysis step is performed. Here, the procedure is slightly different to other kits. Rather than clearing the lysate by centrifugation, a filtration column is included which allows simultaneous clearing and column loading. The filter is designed to reduce shearing of large DNA constructs that can occur via centrifugation, and it can be used even when larger culture volumes are required (e.g. for low copy number plasmids) provided no more than the recommended cell mass is loaded.

Next, the filter and column are washed, then the filter is removed and the column washed again. The DNA is eluted using high salt and pH conditions; therefore, the DNA must be purified by isopropanol precipitation for use in downstream applications. This can be done either by centrifugation (protocol included) or by using the included NucleoBond® Finalizer, a small syringe driven filter-like unit which contains a silica membrane. The DNA is loaded into the Finalizer then washed with ethanol. The DNA can then be eluted with the supplied TRIS buffer or another buffer of choice. The elution step can be repeated using the same aliquot of buffer, which allows complete solubilization of the plasmid. A variety of elution buffer volumes can be used depending on the desired concentration and DNA recovery, and the manual includes tables to help select the most appropriate volume for your needs.

Over the years, we have tried several midiprep kits from different companies. We chose this kit as it has a reasonable price per purification. We have found this kit to require less manipulation and time than other kits, especially as lysate filtration and column loading is done at once. While some other kits use lysate clearing via filtration as well, the NucleoBond® column filter seems both gentle on the DNA (as it is not forced though the filter) and works faster than other gravity fed filters. I have used it to extract both high and low copy number plasmids successfully (4-13 kb) with decent yields. In our lab, this kit is considered the best when working with larger constructs (13-15 kb replicon DNA). I normally use the Finalizer to collect the precipitated DNA, and have found the DNA of a suitable quality for applications such as transfections and restriction digests.