Agarose Gel Electrophoresis

Summary

In this detailed tutorial, Drew Kaulip walks viewers through the process of preparing and running an agarose gel for DNA analysis. From measuring agarose and mixing it with 1X TAE buffer to setting up the casting tray, pouring the gel, and loading DNA samples, every step is covered. The video includes the use of a safer dye, RedSafe, for DNA visualization, and emphasizes the importance of handling the gel carefully. After running the gel electrophoresis, the tutorial highlights how to visualize the results and edit the image for publication. This comprehensive guide is ideal for lab-based DNA analysis experiments.

Takeaways

• 😀 Prepare a 0.8% agarose gel by mixing 0.8 grams of agarose with 100 mL of 1X TAE buffer, ensuring that water is not used.
• 😀 Heat the agarose mixture in a microwave at 70% power for 1-2 minutes until fully dissolved, but allow it to cool before handling.
• 😀 Use masking tape to seal the edges of the casting tray, preventing leaks while pouring the gel.
• 😀 Insert a comb into the gel mixture before pouring to create wells for loading DNA samples.
• 😀 Add 5 microliters of RedSafe to the cooled agarose gel to help visualize the DNA during electrophoresis.
• 😀 Pour the cooled gel into the casting tray, making sure to remove air bubbles with a micropipette tip to avoid interference in the gel.
• 😀 Once the gel solidifies, carefully remove the masking tape and transfer the gel into the electrophoresis unit for running.
• 😀 Use 1X TAE buffer to cover the gel in the electrophoresis unit, ensuring not to disturb the delicate platinum wires inside the chamber.
• 😀 Load DNA samples, including a ladder and plasmid digested with various restriction enzymes, into the wells created by the comb.
• 😀 Run the gel at 120 volts and monitor for the movement of dye and DNA, ensuring the samples travel toward the positive electrode due to the DNA's negative charge.
• 😀 After the run, visualize the gel using a blue LED light box and filter to see DNA bands based on size, and document the results with clear labeling for publication.

Q & A

What is the purpose of making an agarose gel in this experiment?

The agarose gel is made to run DNA samples that have been digested with restriction enzymes. The gel helps visualize and separate DNA fragments based on size during electrophoresis.

Why is it important to use 1x TAE buffer rather than water for making the agarose gel?

1x TAE buffer provides the necessary ions to maintain the pH and conductivity required for proper DNA movement through the gel during electrophoresis. Water would not support the electrophoresis process.

What safety precaution should be taken when handling the heated agarose solution?

Always use heat-resistant gloves when handling the heated agarose solution, as it can be hot enough to burn if touched directly before cooling.

How can you prevent leaks when setting up the gel casting tray?

To prevent leaks, use masking tape to seal the edges of the casting tray before pouring the gel solution. Ensure the tape is pressed firmly and sealed tightly around the edges.

What is RedSafe, and why is it used in agarose gel electrophoresis?

RedSafe is a safer alternative to the toxic ethidium bromide dye. It allows visualization of DNA under UV light without the hazards associated with ethidium bromide. It is added to the gel after it cools.

Why is it necessary to wait for the agarose gel to cool before pouring it into the casting tray?

Allowing the gel to cool ensures it is safe to handle and prevents it from becoming too runny or uneven, which could interfere with proper gel formation and sample loading.

What is the purpose of using a comb in the gel casting process?

The comb is used to create wells in the gel, where the DNA samples will be loaded for electrophoresis. It also helps form the spaces needed for the DNA to migrate during the run.

What happens if you remove the comb too early after pouring the gel?

Removing the comb too early can lead to the formation of air bubbles in the wells, which could prevent the DNA from loading properly, affecting the quality of the electrophoresis results.

Why is it important to ensure that the DNA runs towards the red (positive) electrode during electrophoresis?

DNA is negatively charged, so when an electric field is applied, it migrates towards the positive (red) electrode. This movement allows the separation of DNA fragments based on size.

What can go wrong during DNA loading into the gel wells, and how can it be fixed?

If the DNA is loaded too quickly or if the pipette tip isn't steady, DNA may spill out of the well or not sink properly. To fix this, stabilize the pipette and gently dispense the sample to ensure it stays at the bottom of the well.