Common problems and solutions of agarose gel electrophoresis-Electrophoretic Materials

I.When dissolving agarose in a microwave oven, the gel may boil and overflow from the triangular conical flask. During microwave heating, the gel may boil violently。

1) The total liquid volume should not exceed 50% of the capacity of the conical flask；

2) Set the adhesive solution to medium heat for more than 2%；

3) When the gel solution boils violently, stop heating, remove the conical flask, wear heat-resistant gloves, carefully shake the conical flask, and then heat it again until the gel solution boils until it becomes clear, ensuring complete dissolution of the agarose.

II. The background of the agarose electrophoresis image is blurry

Incomplete dissolution of agarose can cause blurry background in electrophoresis images. The completely dissolved agarose gel solution is clear, and there should be no adhered agarose particles on the inner wall of the triangular conical flask.

III. After heating, the water evaporates. If necessary, add hot distilled water to make up for the original weight and shake well.

1) DNA degradation. Avoid nuclease contamination.

2) Excessive amount of DNA samples. Reduce the amount of DNA sample in gel.

3) Outdated electrophoresis buffer: After multiple uses, the ion strength of the electrophoresis buffer decreases, the pH value increases, and the buffering capacity weakens, thereby affecting the electrophoresis effect. It is recommended to frequently replace the electrophoresis buffer.

4) The electrophoresis conditions are not suitable. During electrophoresis, the voltage should not exceed 20V/cm and the temperature should be less than 30 ℃. For large DNA strands, the temperature should be less than 15 ℃. Check if the electrophoresis buffer used has sufficient buffering capacity.

5) The salt content in the DNA sample is too high. Remove excess salt by ethanol precipitation before swimming.

6) There is protein contamination. Extract and remove proteins before electrophoresis.

7) DNA denaturation. Do not heat before electrophoresis, dilute DNA with 20mM NaCl buffer.

1) Insufficient DNA loading: Increase the amount of DNA loaded.

2) DNA degradation: Avoid nuclease contamination of DNA.

3) DNA goes out of gel: shorten electrophoresis time, reduce voltage, and enhance gel concentration.

4) DNA bands with similar molecular sizes are difficult to distinguish. Increase the electrophoresis time and use the correct gel concentration.

5) DNA denaturation: Do not heat the DNA strand at high temperature before electrophoresis, dilute the DNA with 20mM NaCl buffer.

6) The DNA strand is huge, and conventional gel electrophoresis is not suitable. It was analyzed on pulsed gel electrophoresis.

1) The buffer solution for preparing gel and electrophoresis buffer solution are not prepared at the same time. Use buffer solutions prepared simultaneously. During electrophoresis, the buffer should be 1-2mm above the liquid level.

2) The voltage is too high during electrophoresis. You can use a lower voltage (3V/cm) 15 minutes before electrophoresis, and adjust the voltage after the strip exits the hole.

3) Try to slowly add the sample and wait for it to settle naturally before applying voltage.