Gel recovery/PCR purification gone wrong? Tips to avoid pitfalls and help you recover efficiently!

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 for every 1 mg gel or every 1 μl DNA solution).

2. Insufficient centrifugal force or time is too short:

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

Solution: Ensure that the speed is ≥12,000 rpm during centrifugation. If the centrifuge cannot reach this speed, the centrifugation time needs to be extended accordingly 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, which affects the dissociation of DNA from the membrane.

solve:

• Preferably use the elution buffer (EB) provided by the kit.
• If elution is done with deionized water, the pH value must be adjusted to 7.0-8.5.
• The elution volume was ≥ 30 μl, and it was added dropwise to the center of the silica membrane. It was allowed to 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 DNA template:

Problem: Weak sequencing signal.

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

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

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: Cannot cut or 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.) and set up a positive control to detect enzyme activity.

2. Residual inhibitors in the recovered product:

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

Solution: Ensure that the washing step is centrifuged thoroughly, and if necessary, open the lid and leave it for a short time to allow the residual ethanol to evaporate. If the problem persists, the recovered product can be purified by ethanol precipitation again, and make sure that the volume of the DNA solution does not exceed 10% of the total volume of the enzyme digestion reaction.

4. Electrophoresis abnormality: sample floats 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 (with glycerol/sucrose to increase density and 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), and DNA integrity or purity is compromised.

solve:

• Operate gently, especially when recovering large DNA fragments, to avoid mechanical damage such as violent shaking and blowing that may cause DNA breakage.
• 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 was not rinsed thoroughly:

Problem: Residual inhibitors affect ligation or transformation efficiency.

Solution: Purify the recovered product by ethanol precipitation again.

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, and replace or re-prepare efficient competent cells if necessary.

Recommended efficient recovery tool: StarPure Magnetic Bead Gel Recovery Kit

Want to complete gel recovery/PCR purification in a simpler, more efficient and high-throughput way? Try the StarPure Gel Recovery Kit (Magnetic Beads)!

Core technologies: It uses highly efficient nano-sized silicon hydroxy 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.

Product Performance


Product Recommendations

https://qihengxing.com/products/fs-b6001