Buffer SEP (RNA Extraction)

Absolutely not. Upon adding Buffer A2, the bacterial cells lyse and release genomic DNA. Vigorous vortexing will shear the high-molecular-weight genomic DNA, causing it to mix with the plasmid DNA and making it extremely difficult to remove in downstream steps. Instead, gently invert the tube 4–6 times until the solution becomes clear and viscous.

(1) Imbalanced Bacterial Input: Excessive bacterial culture leads to incomplete cell lysis, while insufficient culture results in an inadequate starting yield. (2) Omission of Ethanol in Wash Buffer: Verify whether anhydrous ethanol was added to Buffer W2 as instructed prior to use. (3) Low Elution Efficiency: Pre-heating the Elution Buffer (Buffer EB) to 50–60 °C can significantly improve efficiency. Apply Buffer EB directly to the center of the column membrane (avoiding the inner wall), let it stand at room temperature for 2 minutes, and then centrifuge to elute.

Buffer A1 can be stored at room temperature before adding RNase A. Once RNase A has been added, store the buffer at 2–8 °C to maintain enzymatic activity, and use it within 6 months.

Perform the gel cutting quickly under UV light and trim as much excess, DNA-free agarose gel as possible to minimize the gel slice volume. A smaller gel slice ensures complete dissolution and higher binding efficiency. Furthermore, excessive gel volume can dilute the solubilization buffer below its optimal ratio, compromising recovery yields.

Set the solubilization temperature to 50–60 °C and invert the tube every 2–3 minutes to promote mixing. If the gel slice is particularly large, extend the heating time or add extra solubilization buffer. Ensure that the gel slice is completely dissolved; any residual agarose will clog the silica membrane of the spin column, severely compromising recovery yield.