DNA gel electrophoresis lab demo

Summary

This video provides a step-by-step guide to performing DNA gel electrophoresis using a mini gel box. The process involves preparing the gel, setting up the gel box with a comb and dams, and carefully pouring the agarose gel. The DNA samples are loaded into wells and separated by size using an electrical current. The video emphasizes key safety precautions, such as wearing gloves and goggles due to the use of ethidium bromide, and explains how to monitor the gel's progress. Finally, it details how to set up and adjust the power supply for optimal results in the lab.

Takeaways

• 😀 Gel electrophoresis is used to separate DNA fragments based on their size, with smaller fragments moving faster through the gel matrix.
• 😀 DNA has a negative charge and will move towards the positive electrode during the electrophoresis process.
• 😀 Proper setup of the gel box is crucial: ensure the box is level using the built-in leveler before pouring the gel.
• 😀 Always wear gloves and goggles when working with the gel as it contains ethidium bromide, a mutagenic substance.
• 😀 The comb used in the gel forms wells where DNA samples are loaded. Make sure there’s a gap between the bottom of the comb and the tray to avoid making holes.
• 😀 The gel should not be poured above the pour line. Ensure it remains below this level to prevent overflow.
• 😀 Once the gel is solidified, carefully remove the comb and rubber dams to avoid damaging the gel.
• 😀 When loading samples, avoid bubbles in the pipette and ensure the pipette tip is positioned properly in the well to prevent sample spillage.
• 😀 Check the orientation of the gel box: the wells should be at the negative electrode end, as DNA moves towards the positive electrode.
• 😀 Once the gel is prepared and samples are loaded, hook up the power supply. Set the voltage to 100-105 volts for proper DNA migration.
• 😀 Monitor the gel box during electrophoresis to ensure the electrical current is flowing (check for bubbles) and that the samples are migrating in the correct direction.

Q & A

What is the purpose of DNA agarose gel electrophoresis?

DNA agarose gel electrophoresis is a technique used to separate different sized fragments of DNA. The DNA, which has a negative charge, moves towards the positive electrode and is separated based on its size, with smaller fragments moving faster through the gel matrix and larger fragments moving slower.

Why do we use gloves and goggles when working with the gel?

We wear gloves and goggles when handling the gel because it contains ethidium bromide, a potential mutagen. The goggles protect against splashes into the eyes, and the gloves prevent skin contact with ethidium bromide.

What does the comb do in the gel electrophoresis setup?

The comb is used to form wells in the agarose gel, which are the spaces where the DNA samples will be loaded. The wells are created by placing the comb in the gel before it hardens.

Why is it important to level the gel box before pouring the agarose gel?

It is important to level the gel box so that the gel will have a flat surface. This ensures that the wells formed by the comb are uniform and that the gel solidifies evenly, preventing uneven DNA separation.

What is the purpose of the spacer in the gel setup?

The spacer is used to ensure that the comb is positioned correctly, maintaining an appropriate gap between the comb and the gel tray. This prevents the comb from piercing the gel or causing holes, which would result in improper sample loading.

What is the purpose of the running buffer in gel electrophoresis?

The running buffer is used to conduct electricity and allow the DNA to migrate through the gel matrix when the power supply is connected. It also helps maintain the pH of the gel during the electrophoresis process.

Why must the wells be positioned at the negative electrode end of the gel box?

The wells must be positioned at the negative electrode end because DNA has a negative charge. When the power is turned on, the DNA will move toward the positive electrode, so the samples need to be placed at the negative end to migrate in the correct direction.

How do you load samples into the wells of the agarose gel?

To load samples into the wells, you use a micropipette to carefully draw up the DNA sample, then gently lower the pipette tip into the well. After reaching the bottom of the well, you press the plunger to release the sample, ensuring it stays in the well without spilling out.

What is the purpose of the sample buffer in the DNA electrophoresis procedure?

The sample buffer contains a dye to track the DNA migration and glycerol to increase the density of the sample. This ensures the samples sink into the wells and do not float away in the running buffer.

What should you check for before turning on the power supply for the gel electrophoresis?

Before turning on the power supply, you should check that the gel box is properly set up, with the wells positioned at the negative electrode end. Additionally, ensure that the lid is secure, the gel is fully covered with running buffer, and that there are no leaks or gaps around the wells.