Valuable Insights | Always Running into Pits When Doing DNA Gel Extraction? Check Out the Step-by-Step Protocol and FAQs 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.

DNA gel recovery is

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

the

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. With its reliable performance, Aizin Bio-purification column can

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

01 experimental principle analysis

after agarose gel electrophoresis,

PCR amplification products were separated according to their molecular weight and arranged in the gel. Compared with the molecular weight of DNA Marker,

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

the target DNA fragment can be obtained

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

of

02 experimental operation process

① Sample pretreatment and binding buffer are mixed

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

② DNA Adsorption and Preliminary Centrifugation

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

the

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

③ First wash impurities remove

the liquid in the discarded 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. Aizin 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.

④ Secondary washing depth purification

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

the

was then centrifuged again to remove residual Wash Solution.

⑤ DNA elution and preservation

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

The

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

The

Aizin 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.

03 Common Problems In-depth Analysis and Solutions

①

The target product recovery rate is low

The

cause

of the problem▪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.

Causes

of

elution efficiency low

▪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,

make the residual ethanol fully volatile;

▪When adding the Elution Buffer, you must ensure that the buffer drops directly on the membrane.

③ Agarose gel is not completely melted

the

cause

of the problem▪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.

④ The gel cut is too large

. The

reason

is that the agarose gel without the target fragment is not accurately cut off, resulting in too large gel volume and increasing the difficulty of subsequent treatment,

affects DNA recovery and purity.

(on the left is the appropriate size of the block, the right is the oversized block)

the

solution

is to carefully identify the target bands, remove the excess gel as much as possible, reduce the gel volume, and improve the efficiency and purity of DNA recovery.

⑤ The

cause

of exogenous DNA contamination

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 exogenous DNA mixing into the sample.

The

solution

is to ensure that the experiment is carried out in a clean environment, strictly enforce the sterile operation process, and reduce the risk of foreign DNA contamination.

04 Precautions

①

The reagent use specification

should be strictly in accordance with the kit instructions, 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.

② Safety protection measures

Many nucleic acid dyes are potentially toxic, so it is necessary to wear gloves, goggles and other protective equipment during operation,

standard treatment of waste, to avoid environmental pollution and human harm.

05-related product recommendation-Aizin purification column

often encounters unsatisfactory recovery rate and easy contamination of samples in DNA gel recovery experiments,

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 and 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.

Scan code free trial consumables ~

END

Company Profile

About Aizin

Aizu Bio, founded in 2009

is committed to the development and production of cutting-edge biological research in the field of high-quality products

focuses on life science, biological engineering technology and laboratory consumables

adhere to the principle of continuous optimization and quality first, and deliver more trustworthy products to you......

choose Aizu, choose quality