Publish Time: 2025-06-09 Origin: Site
In the field of molecular biology research, experiments such as gene cloning, gene expression analysis, and PCR product verification have high requirements on the purity and integrity of DNA samples. DNA gel recovery is a key technology for obtaining high-purity target DNA fragments. Its standardized operating procedures and selection of experimental consumables have an important impact on the smooth development of subsequent experiments.
Every step from sample pretreatment, DNA adsorption and elution to impurity removal requires not only scientific and rigorous operating procedures, but also high-quality experimental consumables to effectively reduce the risk of sample loss and external contamination. With its reliable performance, Aijin Biopurification Column can play an important role in the DNA gel recovery process and provide experimental support for scientific researchers.
01 Analysis of experimental principles
After agarose gel electrophoresis, the PCR amplification products are separated according to their molecular weight and arranged in the gel. Compared with the molecular weight of the DNA marker, find the gel where the target DNA band is located and cut it out. Heat or use special reagents to melt the gel to dissolve the DNA back into the solution. Then, the target DNA fragment can be obtained through ethanol precipitation or column passing.
02 Experimental operation process
① Sample pretreatment and binding buffer mixing
Transfer the PCR reaction mixture to a 1.5 mL microcentrifuge tube and add Binding Buffer I in a 1:3 ratio.
②DNA adsorption and preliminary centrifugation
Place the purification column in a 2 mL collection tube, transfer the mixture from the previous step to the purification column, place it upright at room temperature for 2 minutes, and then centrifuge at 8000 rpm for 1 minute.
③ First washing to remove impurities
Discard the liquid in the collection tube, add 500 μL Wash Solution to the purification column, centrifuge at 12,000 rpm for 1 minute, discard the liquid in the collection tube, and put the purification column back into the original collection tube. Aijin purification columns can be equipped with high-quality glass fiber membranes, which can efficiently retain DNA during the washing process, have stable performance and good repeatability, ensuring DNA purity.
④ Second washing and deep purification
Add 500 μL Wash Solution to the purification column again, and centrifuge at 12,000 rpm for 1 minute. Discard the liquid in the collection tube and centrifuge again to remove residual Wash Solution.
⑤ DNA elution and storage
Transfer the purification column to a clean 1.5mL microcentrifuge tube, add 30 - 50 μL Elution Buffer to the center of the purification column membrane, and incubate at 50°C for 2 minutes. Centrifuge at 12,000 rpm for 1 minute and freeze the PCR product at -20°C for storage or use immediately. Aijin purification columns can be easily opened and closed with one hand and have strong sealing properties, effectively preventing samples from being contaminated during operation.
03 In-depth analysis and solutions of common problems
① The recovery rate of the target product is low
Cause of problem
▪ The gel-cutting operation under UV light takes too long, causing the nucleic acid to be exposed to UV radiation for a long time, and the molecular structure may change, resulting in loss of function;
▪ If the buffer used during electrophoresis has not been replaced for a long time, the pH value will change, affecting DNA adsorption.
Solution
▪ Optimize the glue cutting process and shorten the UV exposure time;
▪ Change the electrophoresis buffer regularly to ensure that its pH value is within the appropriate range.
② Low elution efficiency
Cause of problem
▪ After the washing step, the residual ethanol is not fully evaporated, hindering the elution of DNA from the purification column membrane;
▪ The Elution Buffer was not dripped onto the membrane accurately, resulting in insufficient DNA dissolution.
Solution
▪ Transfer the solution to the purification column, add Wash Solution and centrifuge twice, then open the cap of the tube containing the purification column and let it stand to allow the residual ethanol to fully evaporate;
▪ When adding Elution Buffer, make sure the buffer drops directly onto the membrane.
③ The agarose gel is not completely melted
Cause of problem
▪ During the sol process in a 55°C water bath, the gel was not fully mixed, resulting in incomplete partial dissolution;
▪ If the volume of the gel block is too large, it will not only reduce the sol efficiency and cause abnormal pH value of the solution, but may also change the color of the solution.
Solution
▪ When dissolving, turn the gel upside down several times to fully melt and mix the gel to ensure that no solid agarose remains. After the gel is dissolved, it will generally appear light yellow or almost colorless;
▪ If the glue block is too large, the amount of sol solution can be increased appropriately until the color of the solution returns to normal.
④ The gel is cut into too large pieces
Cause of problem
The agarose gel that does not contain the target fragment is not accurately excised, resulting in an excessively large gel volume, which increases the difficulty of subsequent processing and affects the DNA recovery rate and purity.
The left side shows a suitably sized glue block, and the right side shows an oversized glue block.
Solution
Carefully identify the target band, remove excess gel as much as possible, reduce the gel volume, and improve DNA recovery efficiency and purity.
⑤ Foreign DNA contamination
Cause of problem
The cleanliness of the experimental environment was not up to standard, and the aseptic operating procedures were not strictly followed during the operation, resulting in foreign DNA being mixed into the samples.
Solution
Ensure that experiments are conducted in a clean environment and strictly implement sterile operating procedures to reduce the risk of exogenous DNA contamination.
04 Things to note
① Instructions for using reagents
Strictly follow the instructions of the kit. For example, whether a certain volume of absolute ethanol needs to be added to the Wash Solution, the volume depends on the reagent requirements of the kit, etc.
② Safety protection measures
Many nucleic acid dyes are potentially toxic. During the operation, you need to wear protective equipment such as gloves and goggles, and handle waste in a standardized manner to avoid environmental pollution and human injury.
05 Related product recommendations-Aijin purification column
When DNA gel recovery experiments often encounter situations such as unsatisfactory recovery rates and easy sample contamination, Aijin purification columns provide reliable support for the smooth conduct of experiments with multiple performance advantages.
▪ Made of imported polymer polypropylene (PP) material that meets USP Class VI standards, no pyrogen, no endotoxin, and no DNase/RNase;
▪ Uniform wall thickness, no tube explosion during high-speed centrifugation, safe and reliable;
▪ Can withstand high-speed centrifugation, the maximum centrifugal force can reach 24000xg;
▪ High-precision mold production, no surface coating, no contamination of samples;
▪ Can be easily opened and closed with one hand and has a tight seal;
▪ Can provide high-quality glass fiber membranes with high extraction yield, stable performance, and good repeatability. It is suitable for various experimental operations such as PCR, enzyme digestion, sequencing, and hybridization.