The extracted plasmid has excessive endotoxin levels. Is there a separate remediation method to remove endotoxin?

First, confirm whether the plasmid in your hands, aside from having excessive endotoxin, also has protein and RNA contamination. Ordinary midiprep kits often show elevated levels of all three, and removing endotoxin alone may still leave macrophages in a poor state, so after processing it is best to also measure A260/A280 and run gel electrophoresis. Before attempting remediation of the 300 µg sample, do not use it all up; reserve 10–20 µg as a control. Use endotoxin-free water or Tris for elution, and the collection tubes must also be treated; in many cases, low recovery is actually caused by introducing new endotoxin through ordinary EP tubes or water in the final step. If it is still above 0.1 after treatment, you can pass it through the column once more, but each repetition will result in a 10–20% loss, so you should expect that.

The plasmid has been purified to remove endotoxins. I have used Thermo's high-capacity endotoxin removal resin and domestic resins, and handling 300 µg of plasmid is not a problem. The procedure is to first activate and equilibrate the resin, then mix it with the plasmid solution at a volume ratio of approximately 1:5 to 1:10, shake gently at room temperature for 30 min, and collect the filtrate by centrifugation or by passing through an empty column. The recovery rate is affected by the plasmid concentration; I generally achieve 70–85%. If the initial level is 2 EU/µg, a single treatment may reduce it to 0.1–0.5 EU/µg, but for macrophages it is best to perform two rounds or increase the amount of resin. The total handling time, including detection, is about 1.5–2 h. Note that all tubes and pipette tips that come into contact must be endotoxin-free.

If you want speed and do not want to pack the resin yourself, you can buy ready-to-use endotoxin removal spin columns, such as Pierce or GenScript mini columns. For a 300 µg sample, calculate according to the column binding capacity; generally, the amount processed per run is limited. If the plasmid concentration is high, dilute it first; otherwise, the column may clog easily or recovery may decrease. The advantage of ready-to-use columns is the short handling time, about 20–40 min per run, but they are more expensive than resin. You can first buy the smallest pack and try it once; if the effect is not sufficient, then decide whether to use a larger pack. Before and after processing, be sure to quantify with LAL reagent, and do not judge only by inflammatory response, because macrophages are also very sensitive to low levels of LPS. It is recommended to reduce the target to below 0.1 EU/µg.

If you don't want to buy dedicated columns, you can use Triton X-114 liquid-phase extraction as an emergency measure. After adjusting the plasmid solution to a certain volume, add Triton X-114 to a final concentration of about 1%, mix on ice, then phase-separate at 37°C. After centrifugation, take the upper DNA solution, usually repeating 1–2 times. This method is relatively effective for endotoxin removal, but residual Triton affects macrophages, so it must finally be exchanged into buffer by ethanol precipitation or spin column, then eluted with endotoxin-free water. At a 300 µg scale, recovery is roughly 60–80%, and the time is about 1–1.5 h. I suggest first testing the conditions with a small amount rather than processing all samples at once, to avoid poor recovery and having to re-extract.