Date Published: November 18, 2009
Publisher: Public Library of Science
Author(s): Yun-Cheol Kim, Sherie L. Morrison, Carles Lalueza-Fox. http://doi.org/10.1371/journal.pone.0007750
Abstract: Purity, yield, speed and cost are important considerations in plasmid purification, but it is difficult to achieve all of these at the same time. Currently, there are many protocols and kits for DNA purification, however none maximize all four considerations.
Partial Text: Traditional plasmid DNA purification methods all have some limitations. Some are fast and allow isolation of nucleic acids within an hour, but speed usually comes at the price of reduced yield and/or purity, . Although cesium chloride (CsCl) plasmid purification produces high yield and purity, , it requires extended periods (6 to 24 ours) of ultracentrifugation and the removal of CsCl and ethidium bromide is tedious and generates toxic by-products. Many commercial DNA purification kits including QIAEX II Gel Extraction Kit (Qiagen, Valencia, CA) have been developed based on the fact that DNA binds to glass milk and diatomaceous earths in the presence of chaotropic agents, . Even though these kits are efficient, shearing forces due to fine particles may cause DNA breakage. Use of NaI (Geneclean Kit (Qbiogene, Irvine, CA)), which tends to oxidize over time, can lead to very poor DNA quality or quantity. Although glass filters have been used for small scale, high throughput plasmid purification of plasmid templates suitable for sequencing using PCR, the quantity and the quality of the plasmid purified by these methods may not be suitable for many other applications , . Purification methods based on the fact that the large anion, DNA, can efficiently bind to positively charged resins provide high yield, however there is often contamination with genomic DNA. Although customized anion exchange resins provide efficient DNA purification, they are only available as high priced commercial kits available from vendors including Qiagen and Mackerey & Nagel.
In order to develop a rapid and cost effective method for plasmid purification, we have used glass filters as a DNA binding matrix because of their low price, availability and convenience (Figure.1). Glass filters are sold both as a disc and as a pre-made syringe filter. Although for convenience we primarily used the ready-made glass syringe filters, we found that filter discs placed in a holder yield the same result. Therefore, all results in this paper can be applied to both lab assembled and ready-made syringe filters. Initially, we investigated the DNA binding capacity of glass filters with 0.7 um and 1 um pore size and found that more plasmid was isolated using the 30 mm glass filter with a 0.7 um pore-size (Data not shown). Therefore all subsequent experiments were done using 30 mm glass filters with a 0.7 um pore-size.
Purification of plasmid DNA from E.coli can be tedious and time consuming, but is critical for many experimental procedures. Although it can be simple and easy to use commercial kits for plasmid purification, most kits are expensive. The most widely used kits, including Qiagen-Maxi-prep kits, utilize anion exchange resins to capture plasmid DNA from crude bacterial lysates. Because the flow rate of the cell lysate through the column depends on gravity, it can take 1 to 1.5 hours for the whole procedure.