Abstract
Silica columns from PCR purification and gel extraction kits are widely used in laboratories worldwide to assist in gene cloning. However, the use of these columns can generate plastic waste that has an environmental impact due to their one-off design and massive consumption. Thus, it is important to develop a novel method that can reduce the utilization of silica columns but not affect research efficiency. In this study, various chemical and nonchemical reagents were used to eliminate residual DNA within used columns from PCR purification and gel extraction kits. We show that phosphoric acid is the most effective reagent among those tested to remove DNA contamination from used columns. Columns regenerated using 1 M phosphoric acid have a DNA purification capability that is comparable to that of fresh columns. We demonstrate that silica columns can be regenerated and reused a minimum of five times. The lab-made buffers are compatible with the regenerated columns for DNA purification, and DNA that is prepared with the regenerated columns can be used for gene cloning without affecting the gene cloning efficiency. Thus, the use of this novel method greatly reduces the production of laboratory waste and benefits numerous laboratories worldwide.
Highlights
Molecular cloning is one of the greatest techniques developed in the 20th century[1,2,3] and is used in a range of research areas, including gene replication, recombinant protein preparation, the generation of transgenic organisms, gene function analyses and gene therapy[4,5,6,7,8,9]
The results showed that both PM1and PM2 were able to effectively remove the residual DNA from the used columns, the 0.0122 and 0.0921 pg/μL of residual DNA eluted from the PM1- and PM2-regenerated columns respectively, was significantly higher than that obtained using 1 M phosphoric acid (Fig. 2B)
Previous studies have shown that disposable columns from DNA extraction kits for plasmid preparation do not carry over contaminated DNA from previous experiments[19,20]
Summary
Molecular cloning is one of the greatest techniques developed in the 20th century[1,2,3] and is used in a range of research areas, including gene replication, recombinant protein preparation, the generation of transgenic organisms, gene function analyses and gene therapy[4,5,6,7,8,9]. The goal of this study was to develop a novel method that can reduce the utilization of commercial kits while not affecting research efficiency. A later study demonstrated that used columns from DNA extraction kits can be repeatedly regenerated and utilized for several times without carryover contamination[19], this method is time-consuming. This limitation has been overcome by using a 45-minute approach developed by Tagliavia’s research group[20]. Whether the silica columns from PCR purification kits ( called clean-up kits) and gel extraction kits can be regenerated and reused for gene cloning remains unclear. We show that the quality of DNA prepared with the regenerated columns is sufficient to meet the requirements of gene cloning
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