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How to solve the tailing phenomenon in the electrophoresis process in the electrophoresis equipment?

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Release time:

2021-06-17 19:39

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The tailing phenomenon of electrophoresis in electrophoresis equipment is called smear in English, which is dispersion. The reason is mainly considered from the following two aspects:

 

1. The cause of the PCR product itself:

Often due to too much enzyme or poor enzyme quality, dNTP concentration is too high, Mg2+ concentration is too high,

The annealing temperature is too low and the number of cycles is too much, resulting in too many non-specific PCR products.

 

Countermeasures:

① Reduce the amount of Taq enzyme, or switch to another source of enzyme.

②Reduce the concentration of dNTP.

③Appropriately reduce the Mg2+ concentration.

④ Increase the amount of template, reduce the amount of primers, reduce the number of cycles, and increase the annealing temperature.

 

2. Problems of electrophoresis system:

(1) Contamination of the running buffer TAE or TBE, it is recommended to replace the buffer.

(2) The sample drifts during sample loading. It is recommended to increase the amount of loading buffer and add samples carefully.

(3) The voltage is too high.

(4) Increase the concentration of your glue appropriately. (According to the size of your piece)

(5) Observe whether your marker also has tailing phenomenon, as a control.

 

Causes and countermeasures of PCR tailing and false positive

Smearing phenomenon: The product is in a smear state on the gel.

 

reason:

1. The template is not pure

2. Buffer is not suitable

3. The annealing temperature is low

4. Too much enzyme

5. The concentration of dNTP and Mg2+ is too high

6. Too many cycles

 

Countermeasures:

1. Purification template

2. Replace Buffer

3. Properly increase the annealing temperature

4. Use enzymes in moderation

5. Appropriately reduce the concentration of dNTP and magnesium ions

6. Reduce the number of cycles

 

False positive

Phenomenon: The target amplification product appears in the blank control

reason:

Cross-contamination of target sequence or amplification product

 

Countermeasures:

1. The operation should be careful and gentle to prevent the target sequence from being sucked into the sample gun or spilled out of the centrifuge tube;

2. Except for enzymes and substances that cannot withstand high temperatures, all reagents or equipment should be autoclaved. All centrifuge tubes and sample pipette tips should be used once.

3. It is best to pre-pack various reagents first, and then store at low temperature

Problems in the operation of electrophoresis equipment should be dealt with in a timely manner. There are causes and effects. Finding the right method will definitely solve the electrophoresis tailing phenomenon in the electrophoresis equipment!