Dry Goods Sharing | DNA Gel Recovery Total Step Pit? Operation Steps and FAQ Analysis See Here

In the field of molecular biology, gene cloning, gene expression analysis, PCR product verification and other experiments require high purity and integrity of DNA samples.

as a key technology to obtain high purity target DNA fragments,

of its standardized operation process and the selection of experimental consumables have an important impact on the smooth development of subsequent experiments.

Every link from sample pretreatment, DNA adsorption and elution to impurity removal requires not only scientific and rigorous operation steps, but also high-quality adapted experimental consumables,

to effectively reduce the risk of sample loss and external contamination. A-Gen biological purification column with its reliable performance, can

plays an important role in the DNA gel recovery process and provides experimental support for researchers.

PCR amplification products were separated by molecular weight and arranged in the gel. Compared with DNA Marker molecular weight,

find the gel where the target DNA band is, cut it off, heat or melt the gel with special reagents,

dissolving the DNA back into the solution and then subjecting to ethanol precipitation or passing through the column.

transfer the PCR reaction mixture to a 1.5mL microcentrifuge tube and add Binding Buffer I at a 1:3 ratio.

Place the purification column in a 2mL collection tube and transfer the mixture from the previous step to the purification column,

was placed upright at room temperature for 2 minutes, followed by centrifugation at 8000 rpm for 1 minute.

Discard the liquid in the collection tube, add 500 μL Wash Solution to the purification column, centrifuge at 12000 rpm for 1 minute,

the liquid in the collection tube is discarded, the purification column is returned to the original collection tube. A-Gen purification column can provide assembly of high quality glass fiber membrane,

in the washing process can efficiently retain DNA, stable performance, good repeatability, to ensure the purity of DNA.

again add 500 μL Wash Solution to the purification column and centrifuge at 12000 rpm for 1 min. Discard the liquid in the collection tube,

was then centrifuged again to remove residual Wash Solution.

Transfer the purification column to a clean 1.5mL microcentrifuge tube, add 30 - 50 μL of Elution Buffer in the center of the purification column membrane, and take a warm bath at 50 ℃ for 2 minutes.

was centrifuged at 12000 rpm for 1 minute, and the PCR products were stored frozen at -20°C or immediately tested.

A-Gen purification column can realize easy one-handed opening and closing, and the sealing is strong, which can effectively prevent the sample from being contaminated during operation.

DNA gel recovery01 Experimental Principle Analysis of02 Experimental operation process① Sample pretreatment and binding buffer mixing② DNA Adsorption and Preliminary Centrifugation③ First washing impurity removal④ Secondary washing deep purification⑤ DNA elution and preservation03 Frequently Asked Questions Depth Analysis and Solutions① target product recovery is low Problem causes ▪The glue cutting operation under UV lamp takes too long, resulting in long exposure of nucleic acid to UV radiation, the molecular structure of the may change, thus losing its function;▪The buffer used in electrophoresis has not been replaced for a long time, and the pH value changes, which affects DNA adsorption.Solution ▪Optimize the cutting process and shorten the UV irradiation time;▪Change the running buffer periodically to ensure that the pH is in the proper range.
② Low elution efficiency Problem causes ▪After the washing step, the residual ethanol is not sufficiently volatilized, which hinders the elution of DNA from the purification column membrane;▪The Elution Buffer was not accurately dropped on the membrane, resulting in insufficient DNA dissolution.Solution ▪After the solution is transferred to the purification column, added to the Wash Solution and centrifuged twice, the tube cover equipped with the purification column needs to be opened and allowed to stand,volatilize the residual ethanol;▪When adding the Elution Buffer, you must ensure that the buffer drops directly on the membrane.
③ Agarose gel not completely melted Problem causes ▪In the process of 55 ℃ water bath sol, the gel was not fully mixed, resulting in incomplete local dissolution;▪Excessive volume of the gel block will not only reduce the efficiency of the sol, but also cause the pH value of the solution to be abnormal,may also change the color of the solution.Solution ▪When the sol is turned upside down many times, the gel is fully melted and mixed to ensure that there is no solid agarose residue. After the gel is dissolved, under normal circumstances,will appear pale yellow or almost colorless;▪If the glue block is too large, the amount of sol can be appropriately increased until the color of the solution returns to normal.
The gel cut piece is too large Problem causes the agarose gel that does not contain the target fragment is not precisely excised, resulting in excessive gel volume and increasing the difficulty of subsequent processing,affects DNA recovery and purity.(on the left is the appropriate size of the block, the right is the oversized block)Solution carefully identify the target band, as far as possible to remove the excess gel, reduce the gel volume, improve the efficiency of DNA recovery and purity.
Exogenous DNA contamination Problem causes The cleanliness of the experimental environment is not up to standard, and the operation process does not strictly follow the aseptic operation specifications, resulting in the mixing of exogenous DNA into the sample.Solution ensure that the experiment is carried out in a clean environment, strictly implement the sterile operation process, reduce the risk of foreign DNA contamination.
Considerations for 04① Reagent usage specification should strictly follow the instructions of the kit, such as whether a certain volume of anhydrous ethanol needs to be added to the Wash Solution, the number of volumes depends on the reagent requirements of the kit, etc.Safety protection measures nucleic acid dyes are often potentially toxic, and protective equipment such as gloves and goggles should be worn during operation,standard treatment of waste, to avoid environmental pollution and human harm.
05 Related Product Recommendation-Aizin Purified ColumnIn DNA gel recovery experiments, the recovery rate is often unsatisfactory and the sample is susceptible to contamination,Aizin purification column with a number of performance advantages, to provide reliable support for the smooth development of the experiment.▪USP Class VI standard imported polymer polypropylene (PP) material, non-pyrogenic,No endotoxin, DNase/RNase;▪Uniform wall thickness, high-speed centrifugal tube does not burst, safe and reliable;▪Can withstand high-speed centrifugation, the maximum centrifugal force can reach 24000xg;▪High-precision mold production, no surface coating, will not pollute the sample;▪Can be easily opened and closed with one hand, tightly sealed;▪Can provide the assembly of high quality glass fiber membrane, high extraction rate, stable performance, good repeatability,suitable for PCR, enzyme digestion, sequencing, hybridization and other experimental operations.
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