Agarose Gel Electrophoresis

Summary

This instructional video guides viewers through the process of agarose gel electrophoresis to separate DNA samples. It details the setup of a mini-sub cell with electrodes, the alignment of gels, and the addition of running buffer. The script instructs on loading DNA samples into the gel wells, ensuring proper placement and technique to avoid contamination. It also covers connecting the gel to a power supply, setting the voltage, and observing the DNA migration towards the positive electrode, providing a clear and concise tutorial for DNA analysis.

Takeaways

• 🧬 Use a mini-sub cell for agarose gel electrophoresis, which has electrode wires for electric current.
• 🔌 DNA fragments move from the negative (black) electrode to the positive (red) electrode due to their negative charge.
• 🧪 Align the wells of the gel closest to the negative electrode before loading the samples.
• 💧 Add electrophoresis running buffer to the reservoirs and wells until they are covered by at least 2mm of buffer.
• 📝 Follow the lab protocol for loading samples in the correct order according to assigned lanes.
• 💉 Use an adjustable micropipette to obtain and load DNA samples, ensuring the tip is close to the bottom of the sample tube.
• 🚫 Keep the pipette tip perpendicular to the wells to reduce the risk of puncturing them.
• ⚠️ After loading, avoid bumping or moving the gel chamber to prevent sample spillage.
• 🔌 Connect the electrodes to the power supply, ensuring the correct color-coded connections (black to black, red to red).
• ⏱️ Set the power supply to the appropriate constant voltage and timer as specified in the lab protocol.
• 📊 Observe the migration of DNA samples from the wells into the gel, moving towards the positive electrode.

Q & A

What is the purpose of using a mini-sub cell in agarose gel electrophoresis?

A mini-sub cell is used to perform agarose gel electrophoresis, which allows the separation of DNA fragments in the samples by applying an electric current through the gel.

How does the electric current affect the movement of DNA fragments in the gel?

DNA is negatively charged and will move from the negative electrode through the gel towards the positive electrode due to the electric current.

What should be the orientation of the agarose gel in relation to the electrodes?

The wells of the agarose gel should be closest to the negative or black electrode for optimal DNA fragment separation.

How should the electrophoresis running buffer be added to the gel chamber?

The electrophoresis running buffer should be added to the reservoirs at each end of the gel chamber, and at least two millimeters of buffer should cover the wells in the gel.

What is the correct order for loading DNA samples into the wells of the gel?

Samples should be loaded in the correct order according to the lanes they are assigned to, as specified by the lab protocol.

How should a DNA sample be taken from a microtube using an adjustable micropipette?

To obtain a DNA sample, slowly depress the plunger on the adjustable micropipette to the first or soft stop, then insert the tip into the microtube with the DNA sample, releasing the plunger button to draw the sample into the pipette.

What is the proper technique for loading samples into the wells without damaging the gel?

The pipette tip should be kept perpendicular to the row of wells, and the tip should be lowered until it breaks the surface of the buffer just above or just inside the well to reduce the risk of puncturing the wells.

Why is it important to avoid bumping or moving the gel chamber after loading the samples?

Bumping or moving the gel chamber after loading samples might result in the samples spilling into adjacent wells, which can interfere with the electrophoresis process.

How should the lid of the gel chamber be positioned when connecting it to the power supply?

The lid should be placed on the gel chamber with the terminals correctly positioned to match the electrodes on the gel chamber, with black to black and red to red.

What should be checked before starting the electrophoresis process?

Before starting, ensure that the electrodes are correctly connected to the power supply, the correct constant voltage is set according to the lab protocol, and if available, the timer is set for the proper time.

What is the expected visual indication of the electrophoresis process being initiated?

Upon starting the electrophoresis, bubbles should be visible coming from the wires at each end of the gel box, with fewer bubbles at the positive or red electrode.