Electrophoresis: How to Run an Agarose Gel | William Armour

Summary

This video demonstrates the process of performing agarose gel electrophoresis to analyze PCR samples. The technique separates DNA molecules by size and charge, with samples running through a gel matrix under an electric current. The video walks through preparing the gel, adding samples, and running the electrophoresis. After the gel has set and the samples are loaded, an electric current is applied, and the gel is analyzed under UV light to visualize the DNA bands. The presence and size of these bands indicate whether the PCR amplification was successful, showing homozygous or heterozygous DNA patterns.

Takeaways

• 😀 Agarose gel electrophoresis is a method used to separate DNA molecules based on their charge and size.
• 😀 The process involves running an electric current through a gel to move DNA samples, which are visualized under UV light.
• 😀 Agarose gel is prepared by mixing agarose powder with Tris borate EDTA buffer and heating it to dissolve the agarose.
• 😀 Ethidium bromide is added to the gel solution to bind with DNA, enabling visualization of the DNA bands under UV light.
• 😀 The gel is poured into a gel former, and a comb is used to create wells for loading the DNA samples.
• 😀 After the gel sets, the wells are ready to receive the samples, and the gel is placed in an electrophoresis tank.
• 😀 The electrophoresis tank is filled with buffer solution, and an electric current is applied to move the DNA samples through the gel.
• 😀 The size of the DNA fragments affects how far they travel: smaller fragments move faster through the gel, while larger fragments move slower.
• 😀 The gel is run for 30 minutes, after which it is removed, and the DNA bands are visualized under UV light, with bands fluorescing due to the ethidium bromide.
• 😀 A reference ladder is used to estimate the size of the DNA bands, providing a comparison for analyzing the results.
• 😀 The bands that appear on the gel indicate successful PCR amplification, with different band patterns suggesting homozygosity or heterozygosity in the samples.

Q & A

What is the purpose of agarose gel electrophoresis?

Agarose gel electrophoresis is used to separate DNA molecules based on their size and charge by applying an electric current through a gel, allowing the molecules to move at different rates.

What is the role of ethidium bromide in the gel preparation?

Ethidium bromide binds to DNA, enabling the visualization of DNA fragments under UV light after the electrophoresis process.

How is the agarose gel prepared?

To prepare the agarose gel, 1g of agarose is mixed with 100 mL of Tris-borate-EDTA (TBE) buffer. The solution is heated to dissolve the agarose and then ethidium bromide is added before pouring the gel into the former.

What is the purpose of using a reference DNA ladder in the experiment?

The reference DNA ladder, which contains known DNA fragment sizes, is used to compare and estimate the size of the DNA fragments in the sample.

Why is it important to prevent air bubbles when preparing the agarose gel?

Air bubbles in the gel can interfere with the movement of the DNA samples, leading to inaccurate results or distorted bands during electrophoresis.

What happens during electrophoresis once the current is applied?

During electrophoresis, DNA fragments move through the gel, with smaller fragments traveling faster and further than larger ones, allowing them to be separated based on size.

Why are fresh pipette tips used for each sample when loading the gel?

Fresh pipette tips are used for each sample to avoid cross-contamination between samples, ensuring accurate and reliable results.

How can the success of PCR amplification be checked using agarose gel electrophoresis?

The presence of distinct bands in the gel after electrophoresis indicates that the PCR amplification was successful. A band in the expected size range confirms successful amplification of the DNA target.

What do the different band patterns in the gel indicate about the DNA samples?

Different band patterns indicate the presence of different genotypes. For example, homozygous samples will show one band, while heterozygous samples will show two bands representing different DNA fragment sizes.

What is the role of the TBE buffer during electrophoresis?

The TBE buffer provides an electrically conductive solution that allows the DNA to move through the agarose gel when the electric current is applied, while also helping to maintain the pH and stability of the gel during the process.