Entmystifizierung der Nukleinsäure-Agarose-Gelelektrophorese

In molekularbiologischen Experimenten, verifying the quality of nucleic acids extracted by magnetic bead method through agarose gel electrophoresis (gel running) is a crucial step. Jedoch, beginners often encounter some confusing electrophoresis phenomena. What information lies behind these abnormal phenomena? Then how can it be prevented?

1. Band tailing: A warning of nucleic acid degradation

When the band shows continuous smear-like tailing from top to bottom, it usually indicates that the nucleic acid has degraded. This might be because:

1. Degradation of the sample itself (failure to handle it promptly after collection or repeated freezing and thawing

2. Nuclease contamination is introduced during the operation process

3. The storage time after cracking is too long

4. Excessive mechanical shearing force (overly vigorous blowing and striking)

Preventive measures: Use fresh samples; Ensure that the experimental environment is clean and free from pollution; The operation process is gentle. After extraction is completed, test it promptly or store it at -80℃.

2. Band blurring: A signal indicating residual impurities

If the band diffusion is blurred and the boundary is not clear, the most common reason is:

1. Residual salt ions (incomplete washing

2. Protein or polysaccharide contamination

3. Ethanol residue (affecting electrophoretic mobility

Preventive measures: Ensure that all waste liquid is thoroughly sucked up after the last wash. Allow the magnetic beads to dry moderately (the surface should be dull but not cracked); Für komplexe Proben, the number of washes can be increased.

3. Smiling bands: Improper electrophoresis conditions

The two ends of the band curve upward in a “smile” shape, which is usually related to the electrophoresis conditions:

1. Excessive electric field intensity (too high voltage)

2. Uneven gel cooling

3. The ionic strength of the electrophoresis buffer is inappropriate or it has been reused too many times

Preventive measures: Appropriately reduce the electrophoresis voltage; Use freshly prepared electrophoresis buffer; Make sure the electrophoresis tank is equipped with an appropriate cooling device.

4. Other frequently asked questions

No band: It may be due to magnetic bead loss, insufficient elution or insufficient sample size

Abnormal bright spots: It may be RNA contamination (in DNA samples) or genomic DNA contamination (in RNA samples)

Professional advice: Positive and negative controls should be set up for each experiment. This helps to quickly determine whether the problem lies in the sample itself or the extraction process. Gleichzeitig, remember that glue running is only the first step in quality inspection. For downstream experiments such as qPCR and sequencing, it is recommended to use Nanodrop or Qubit for precise quantification.

Understanding the causes and solutions of these common problems can not only help you achieve beautiful electrophoresis results, but also be the key to ensuring the success of downstream experiments. Good experimental habits and meticulous technical operations are the foundation for obtaining high-quality nucleic acid samples.

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Shanghai Lingjun Biotechnology Co., Ltd.wurde gegründet in 2016 Das ist ein professioneller Hersteller von biomagnetischen Materialien und Nukleinsäure-Extraktionsreagenzien.

Wir verfügen über umfangreiche Erfahrung in der Extraktion und Reinigung von Nukleinsäuren, Proteinreinigung, Zelltrennung, Chemilumineszenz, und anderen technischen Bereichen.

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