For those of you who have run agarose gel electrophoresis, have you often encountered the phenomenon of electrophoresis tailing? I believe many people are nodding like crazy. There are plasmid tailings, DNA tailings, even PCR tailings, tailings are the most common. Forget about verification, the key is that the pictures don’t look good when you take them, and posting articles or something like that affects your quality too much. Next, I will tell you about why agarose gel electrophoresis tails based on my own experience. If there are any shortcomings, I hope you can comment and make up for them (I must be my teacher when we are together)! !

Why does agarose gel electrophoresis smear?

1. There are many factors that cause tailing of PCR products, which can be summarized into one factor: excessive non-specific amplification. The reasons may be:

(1) The template is impure: purify the template

(2) The Buffer is inappropriate: replace the Buffer

(3) The annealing temperature is too low : Increase the annealing temperature appropriately

(4) Too much enzyme: use an appropriate amount of enzyme, or replace it with another enzyme

(5) The concentration of dNTP and Mg2 is too high: reduce the dNTP appropriately and the concentration of magnesium ions

(6) Too many cycles: reduce the number of cycles

(7) The amount of template is too little or the amount of primers is too much: increase the amount of template and reduce the amount of primers

2. This phenomenon often occurs when extracting plasmids. It may be that the sample concentration is too high. This situation is simple, just dilute it.

3. Tailing often occurs when extracting genomic DNA. The most likely reason is that the extracted DNA is doped with protein, and the protein will drag the swimming of DNA. Therefore, we must pay attention to the operation of extracting genes to reduce the appearance of impurities such as proteins.

4. The sample is broken or degraded

5. RNA is present: The piece in front of the DNA is usually uncleaned RNA. After purification and RNAse treatment, it is gone

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6. Problems with the electrophoresis system: Observe whether your marker also has tailing phenomenon, as a control

(1) Contamination of the electrophoresis buffer TAE or TBE:. It is recommended to change the buffer.

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

(3) The voltage is too high: lower the voltage appropriately

(4) According to the size of the nucleic acid fragment, increase the gel concentration appropriately. The choice of gel concentration depends on the size of the DNA molecules. The gel concentration required to separate DNA fragments less than 0.5kb is 1.2-1.5,

The gel concentration required to separate DNA molecules larger than 10kb is 0.3-0.7, and the DNA fragment size is between the two. The glue concentration is 0.8-1.0. /s/QZbboqNBDce2QFPD8JxFCw