Question 1

I am using the kit for the first time and noticed that the wash buffer (Buffer W2) smells like alcohol. Do I still need to add ethanol?

Accepted Answer

It is important to distinguish between the two. Wash buffers (such as Buffer W2) in standard extraction kits are typically supplied as concentrated solutions. Before first use, you must strictly follow the instructions on the bottle label by adding the specified volume of anhydrous ethanol and mixing thoroughly. Failing to add ethanol will lead to elution failure, resulting in no plasmid yield.

Question 2

After adding Buffer A2 (Lysis Buffer), the bacterial suspension becomes viscous. Can I mix it by vigorous vortexing?

Accepted Answer

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.

Question 3

What could be the reasons for a low plasmid yield?

Accepted Answer

(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.

Question 4

How should Buffer A1 (containing RNase A) be stored?

Accepted Answer

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.

Question 5

What tips can help improve recovery efficiency during gel extraction?

Accepted Answer

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.

Question 6

What should I do if the gel slice remains undissolved after heating in the solubilization buffer for a long time?

Accepted Answer

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.

Question 7

The recovered DNA was used for sequencing, but the results indicate residual ethanol. How can this be resolved?

Accepted Answer

Residual ethanol usually occurs due to incomplete centrifugation following the wash step. After discarding the Buffer WG2 wash solution, place the empty spin column back into the centrifuge and spin it at maximum speed for 2 minutes. Then, leave the column open at room temperature for 1–2 minutes to allow any remaining ethanol to evaporate completely before proceeding with elution.

Question 8

Can this kit remove primer dimers?

Accepted Answer

Yes. Utilizing specific silica membrane adsorption technology, Aijin Biotech's PCR Purification Kit effectively recovers DNA fragments ranging from 100 bp to 10 kb. Impurities smaller than 100 bp—such as primer dimers and single nucleotides—flow through the membrane and are eliminated, yielding high-purity PCR products.

Question 9

Why does the purified DNA show a bright band on agarose gel electrophoresis, but the measured concentration is very low?

Accepted Answer

This is often caused by an excessively large elution volume, which dilutes the DNA and results in a low concentration reading despite a high total yield. If your downstream applications require a higher concentration, reduce the elution volume (e.g., to 30–50 $\mu	ext{L}$). Additionally, ensure that the elution buffer is applied directly to the center of the membrane to achieve maximum recovery and concentration in a smaller volume.

Product List	A-GEN Number	Spec	Product Description	Packaging Specification
Product List	A-GEN Number	Spec	Product Description	Packaging	Mini Pack	Pieces/Box
	AG1206	50 tests/box	Efficiently lyse bacterial cells to purify endotoxin-free plasmid DNA in midiprep scale, meeting the requirements of mid-scale cell transfection and downstream molecular biology applications.	50T/box	50T	-

Endo-free Plasmid DNA Double Mini Kit

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