DETEKSI AGEN PENYAKIT IKAN DENGAN POLYMERASE CHAIN REACTION (PCR) - part III

Summary

This video tutorial provides a step-by-step guide on performing agarose gel electrophoresis for DNA analysis. It starts with the preparation of the agarose gel, followed by the creation of the electrophoresis setup, including loading DNA samples and markers into the gel. The video emphasizes the importance of proper sample handling and electrophoresis conditions, such as applying the correct voltage and duration. After running the samples, the gel is stained and observed under UV light to visualize the DNA bands, which are then documented. Throughout, the video blends instructional content with casual expressions and background music for an engaging learning experience.

Takeaways

• 😀 The process of preparing an agarose gel for electrophoresis begins by weighing agarose and adding it to an Erlenmeyer flask.
• 😀 Buffer solution is added to the agarose, which is then heated until it boils and cooled until solidified.
• 😀 Once the agarose gel has solidified, it is poured into a tray and allowed to cool completely.
• 😀 The gel is placed into an electrophoresis chamber, and a buffer solution is added until the tray is submerged.
• 😀 DNA samples are loaded into the wells of the gel. A micro-pipette is used to add 3 microliters of the sample into each well.
• 😀 A DNA marker (5 microliters) is added to the gel to help identify the size of DNA fragments after electrophoresis.
• 😀 PCR products are also loaded into the wells, including negative and positive control samples for comparison.
• 😀 The electrophoresis machine is set to 120 volts for about 20 minutes, with the wells positioned at the negative pole (since DNA is negatively charged).
• 😀 The DNA samples move through the gel, and after the electrophoresis run, the machine is turned off.
• 😀 The gel is then soaked in water for about 10 minutes and visualized under UV light using a transilluminator for documentation of the DNA bands.

Q & A

What is the first step in preparing an agarose gel for electrophoresis?

The first step is to weigh the agarose and place it into an Erlenmeyer flask.

How is the agarose gel mixture homogenized?

The agarose is mixed with a buffer solution and then homogenized by stirring to ensure even distribution.

What should be done after the agarose solution is prepared?

The agarose solution is heated until it boils and then allowed to cool down until it solidifies.

What is the purpose of the electroforesis chamber in the procedure?

The electrophoresis chamber is used to run the agarose gel with the DNA samples placed into wells for separation based on size.

How is the gel placed in the electrophoresis chamber?

The gel is placed along with the tray into the electrophoresis chamber, and buffer solution is added until the tray is submerged.

How are the DNA samples prepared for loading onto the gel?

The DNA samples are mixed with a loading dye and pipetted into the wells of the agarose gel.

What is the role of the DNA markers in electrophoresis?

The DNA markers are added to the gel to serve as a reference for determining the size of the DNA fragments in the samples.

What is the typical voltage setting used for electrophoresis in this procedure?

The electrophoresis machine is set to 120 volts for about 20 minutes.

How do you know when the electrophoresis run is complete?

The electrophoresis run is considered complete when the DNA has migrated approximately three to four gel lanes.

What is done after the electrophoresis run is completed?

After the run, the power is turned off, the gel is removed, and it is stained with a DNA stain to visualize the bands under a UV transilluminator for documentation.