Why does the plasmid DNA extraction kit require two washings with two types of wash buffers (W1 and Wash Buffer) while competitors kits require one washing? "

W1 Buffer is used to remove protein residues and degraded RNA residues on the membrane and the Wash Buffer is used to remove salt residues on the membrane. When a small volume of bacterial culture (less than 3 ml) is used, the lysate is usually not rich in protein contaminants so washing with only the Wash Buffer is already enough to result in plasmid pure enough for DNA sequencing and other applications. As one may notice, when a kit only includes one wash buffer, it only allows purification of plasmid DNA from a culture with a volume of less than 3 ml. This is because one wash buffer is not enough to remove contaminants from a higher volume of culture. This type of product only allows for isolation of high copy plasmid as a small volume of culture is used. It cannot be used to isolate low copy plasmid as a higher volume of culture is required. Moreover, the drawback of using only one wash buffer is that it cannot remove degraded RNA bound to the membrane. Removal of RNA existing in the bacterial cells is achieved by degrading RNA released from cells by RNase added in PD1 Buffer. Degraded RNA does not bind well to the membrane in the presence of chaotropic salts, thus degraded RNA is washed off with the wash buffer which contains chaotropic salts, whereas plasmid DNA is still bound to the membrane and is then eluted without RNA contamination. The single wash buffer provided in other kits does not contain chaotropic salts as our Wash buffer does, thus it is not able to remove degraded RNA bound to the column. In this case, degraded RNA will be co-eluted with plasmid DNA. Since RNA is degraded, the user is unable to see it by agarose gel electrophoresis analysis. Though degraded RNA does not affect restriction digestion or sequencing reactions, the presence of the ribo-oligonucleotides interferes with some applications such as digestion of plasmid with BAL 31 or labeling of the 5’ termini of restriction enzyme fragments of the plasmid with bacteriophage T4 polynucleotide kinase. Further, the presence of degraded RNA leads to a false high OD260 of the plasmid eluate (degraded RNA also absorbs light at wavelength of 260 nm), thus misleading the users, who assume that a high plasmid yield is obtained. The presence of degraded RNA in the plasmid DNA solution can be evidenced by OD260/OD280 ratio higher than 1.8. The use of two wash buffers provided in Geneaid's kit solves these issues.