Gel recovery/PCR purification gone wrong? Avoid these pitfalls and get efficient recovery!

1. Low recovery rate: Where does the DNA go?

1. Improper amount of membrane binding solution (MB):

Problem: Wrong liquid addition ratio affects DNA binding efficiency.

Solution: Add in a strict 1:1 ratio (i.e., add 1 μl MB to every 1 mg of gel or every 1 μl of DNA solution).

2. Insufficient centrifugal force or time is too short:

Problem: DNA is not adequately bound to the silica membrane.

Solution: Ensure the centrifuge speed is ≥12,000 rpm. If the centrifuge cannot reach this speed, the centrifugation time should be extended to ensure that the solution completely passes through the silica membrane.

3. Membrane rinse solution (MW) without ethanol added:

Problem: Ethanol was not added to the MW solution as instructed, resulting in poor washing results and impurities remaining.

Solution: Before use, make sure that ethanol has been added to the MW bottle in proportion.

4. Poor elution conditions:

Problem: The pH value of the elution buffer is inappropriate or the volume is too small, affecting the dissociation of DNA from the membrane.

solve:

• It is preferred to use the elution buffer (EB) provided by the kit.
• If elution is done with deionized water, the pH must be adjusted to 7.0-8.5.
• The elution volume should be ≥30 μl, which should be added dropwise to the center of the silica gel membrane. Let it stand for 1 minute to allow the membrane to be fully infiltrated before centrifugation.

2. Poor sequencing results of recovered products: weak signal or large interference

1. Insufficient amount of DNA template:

Problem: Weak sequencing signal.

Solution: Increase the amount of DNA used in the sequencing reaction. If the recovered product concentration is too low, concentrate it via ethanol precipitation before use.

2. The amount of DNA template is too high or inhibitors are present:

Problem: Excess DNA or impurities interfering with the sequencing reaction.

• Appropriately reduce the amount of sequencing DNA. If necessary, dilute the recovered product with EB or sterile double-distilled water.

3. DNA damage:

Problem: UV irradiation causes DNA damage (e.g., pyrimidine dimers).

Solution: Cut the gel quickly to minimize the exposure time of DNA to UV light.

3. Poor enzymatic cutting effect: unable to cut or not cut cleanly

1. Problems with the endonuclease itself:

Problem: Enzyme inactivation or improper handling.

Solution: Strictly follow the manufacturer's instructions (temperature, buffer, time, etc.). Set up a positive control to test enzyme activity.

2. Inhibitor residues in recovered products:

Problem: The ethanol or salt ions in the membrane rinse solution (MW) are not completely removed, inhibiting enzyme activity.

Solution: Ensure thorough centrifugation during the wash step, and if necessary, briefly open the tube to allow residual ethanol to evaporate. If the problem persists, re-purify the recovered product by ethanol precipitation, ensuring that the volume of the DNA solution does not exceed 10% of the total digestion reaction volume.

4. Electrophoresis abnormalities: sample drifts out or bands are blurred

1. Sample wells are washed out:

Problem: Sample is not dense enough or contains volatile components.

solve:

• Be sure to add loading buffer (contains glycerol/sucrose to increase density and a dye to indicate progress).
• Completely remove residual ethanol (the main component of MW) using the same method as above (centrifuge thoroughly and evaporate with the lid open).

2. Bands are not sharp (smearing, smearing), indicating that DNA integrity or purity is compromised.

solve:

• Operate gently, especially when recovering large DNA fragments, to avoid mechanical damage such as vigorous shaking and pipetting that may cause DNA breakage.
• Strictly prevent cross contamination: use freshly prepared agarose gel and electrophoresis buffer, and keep the gel cutting blade clean.
• Prevent DNA degradation: If the cut gel blocks cannot be recovered immediately, they should be stored at 4°C.

5. Low cloning efficiency: no transformation or high background

1. The adsorption column is not rinsed thoroughly:

Problem: Residual inhibitors affect ligation or transformation efficiency.

Solution: The recovered product was purified again by ethanol precipitation.

2. DNA end damage:

Problem: Exonuclease contamination may be introduced during the recovery process, resulting in terminal deletions.

Solution: Use freshly prepared electrophoresis-related reagents (gel, buffer) and ensure the sterility of the recovery operation as much as possible.

3. Poor efficiency of competent cells:

Problem: The cells themselves have low transformation efficiency.

Solution: Check whether the preparation and storage methods of competent cells are correct. If necessary, replace or re-prepare efficient competent cells.

Recommended efficient recovery tool: StarPure Magnetic Bead Gel Recovery Kit

Want to perform gel extraction/PCR purification more simply, efficiently, and at a high throughput? Try the StarPure Gel Extraction Kit (Magnetic Beads)!

Core technologies: It uses high-efficiency nano-sized silanol magnetic beads and a unique gel melting buffer to rapidly bind DNA.

Operational advantages:

No centrifugation required : Cooperate with magnetic stand/magnetic plate to make the process more friendly.

High throughput & automation compatible ： Easily handle multi-sample processing and easy to automate.

High recovery rate ： Ensure sufficient and high-quality DNA.

Widely applicable ： Suitable for the recovery and purification of PCR products, enzyme digestion products and other DNA fragments separated by agarose gel electrophoresis.

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