Using the same kit to extract bacterial plasmids and genomic DNA, what should I do if the 260/230 ratio is always low?

Plasmid and genomic DNA purification have fundamentally different logics, so a universal kit often ends up serving neither purpose well. If budget is not a major concern, it is recommended to switch to an endotoxin-free plasmid miniprep kit for plasmids—especially for Gram-negative bacteria, which have abundant LPS, as the endotoxin removal step can significantly improve restriction digestion and downstream experiments. For genomic extraction, choose a dedicated bacterial genomic DNA kit that includes proteinase K and RNase digestion, and if necessary, add lysozyme or a lysis buffer designed for Gram-negative bacteria. When selecting, pay attention to the 260/230 ratio; generally, well-purified DNA should be above 1.8–2.0, but do not focus on the ratio alone—run a 0.8% agarose gel to check for RNA contamination and genomic integrity. Poor plasmid digestion efficiency may also be due to residual RNA, so ensure sufficient RNase A is added and given adequate time to act. If yields remain low after switching kits, you can do a final pass through a PCR purification column or dialysis, though this will result in some DNA loss. Try a small package first.

A 260/230 ratio of only 1.5 is most likely due to residual guanidine salts, polysaccharides, or proteins. Don't rush to switch kits yet—strengthen the wash steps of the universal kit: for the first wash after binding, use a wash buffer containing ethanol, and you can increase the volume up to the maximum; after centrifugation, be sure to pour off completely. For the second wash, use 75% ethanol and wash twice, with a 1-minute empty spin each time. Before elution, centrifuge the empty column for 2–3 minutes to allow the membrane or magnetic beads to dry thoroughly, but do not over-dry. If the lysate of Gram-negative bacteria is viscous after lysis, centrifuge first, take the supernatant, and then load it onto the column to avoid clogging the column with lysate. Poor downstream restriction digestion is very likely due to inhibition by residual ethanol or salt. It is recommended to preheat the elution buffer to 55–60°C in advance, and after elution, let it stand at room temperature for 2 minutes before centrifugation. You can elute with nuclease-free water or TE. If the 260/230 ratio is still low after measurement, perform an additional column purification. First, run 3 parallel groups using the same batch of samples to see whether it is an operational issue or the reagent itself.

If you do not want to change brands, you can add a "secondary purification" step after the existing extraction: pass the sample through a commercial PCR purification column or genomic cleanup column again, add 3-5 volumes of binding buffer, and elute as usual; for genomic DNA, this can avoid shearing, and it also helps with plasmids. This method is suitable when the sample size is not large and the budget is sufficient, but it adds 30-40 minutes. When comparing suppliers, ask clearly whether the wash buffer is specifically designed to remove salts and proteins. Some kits have a "high-salt, low-ethanol" wash or dual wash buffers, which are cleaner than general-purpose ones. It is recommended to compare 2-3 vendors and request Gram-negative bacteria extraction data, especially 260/230 and restriction enzyme digestion validation. Do not repeatedly exchange products. First, have the manufacturer's technical support remotely review your operation and Nanodrop curve to confirm that the issue is not instrument baseline or elution buffer background. If the 260/230 of the elution buffer blank is also low, you may need to change the elution buffer. Generally, adding one extra wash can improve it. If it still does not work, then consider switching to an endotoxin-specific kit.

I encountered the same issue as well, but after switching to a silica gel column and adding two additional washes, the ratio improved significantly.

Same question here. When extracting genomic DNA, the 260/230 ratio is always around 1.5. The kit manufacturer says it's normal, but the downstream restriction enzyme digestion efficiency is poor.