Lab Report: Agarose Gel Electrophoresis and DNA Extraction

Abstract

The goal of this laboratory experiment was to perform agarose gel electrophoresis using six different dyes and to extract DNA from wheat bacterium. Agarose gel electrophoresis is a common technique used in molecular biology to separate molecules based on their charge, size, and shape. In this procedure, DNA molecules are moved through an agarose gel matrix using an electric field, and their migration rate is proportional to their size. The extracted DNA was observed as a thick web of strands on a spooling rod.

Through this experiment, we gained valuable insights into the principles and practice of agarose gel electrophoresis and the process of DNA extraction from cells.

Introduction

Agarose gel is a commonly used substance in scientific techniques such as gel electrophoresis and size exclusion chromatography. It is made from purified agarose powder that is boiled in a buffer solution and then cooled to form a gel. Agarose gel electrophoresis is widely used to separate and analyze proteins and DNA molecules based on their size and charge.

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During electrophoresis, an electric field is applied, causing DNA or RNA molecules to migrate through the gel matrix towards the positive pole. Smaller molecules move more quickly through the gel than larger ones, allowing scientists to determine the size of the molecules.

Electrophoresis is essentially a sieving process, separating molecules based on their size, charge, and shape. DNA molecules with larger sizes migrate more slowly through the gel as they become entangled in the matrix, while smaller pieces move faster. The relationship between size and migration rate is generally linear within most of the gel, except for very large fragments.

By altering the concentration of agarose, the matrix can be modified to provide different resolutions. A standard 1% agarose gel can resolve DNA fragments ranging from 0.2 to 30 kb in length.

Agarose is extracted from various species of red marine algae, primarily from the Gelidium genera of seaweed. Agar, derived from the Malay word "agar-agar," meaning jelly, consists of agarose and agaropectin molecules and is used not only in molecular biology but also as a food thickener, laxative, and a medium for growing microorganisms.

Materials and Methods

Materials:

• Agarose gel
• Electrophoresis chamber
• Dyes (six different types)
• Buffer solution
• DNA samples
• Gel loading buffer
• Glycerol
• Spooling rod
• Ethanol
• Pipettes
• Test tubes

Methods:

1. We prepared agarose gel by dissolving agarose powder in a buffer solution, boiling it, and allowing it to cool into a gel matrix.
2. We loaded six different dyes into wells in the agarose gel, which was placed in an electrophoresis chamber containing buffer solution.
3. We applied an electric field to the gel, causing the dyes to migrate through the gel based on their charge, size, and shape.
4. We observed the movement of the dyes and recorded the results.
5. We extracted DNA from wheat bacterium using a spooling rod. As the rod rotated in an aqueous-alcohol interface, DNA attached to the rod.
6. We observed the extracted DNA on the spooling rod.

Experimental Procedure

Agarose gel electrophoresis was conducted using six different dyes, and the results are summarized in the table below:

In the DNA extraction experiment, the appearance of the extracted DNA was that of a thick web of strands on the spooling rod, with a rusty white color resembling wet cotton.

The four types of nucleotides in DNA are adenine, thymine, guanine, and cytosine. Adenine pairs with thymine, while guanine pairs with cytosine. Adenine and guanine are purines, composed of two rings, making them larger bases. Cytosine and thymine are pyrimidines, with only one ring, making them smaller than purines. A nucleotide consists of a 5-carbon sugar, a phosphate group, and a nitrogenous base.

Results

The agarose gel electrophoresis experiment showed that dyes with negative charges migrated faster, while those with positive charges migrated slower. Dye 5, which was neutral, did not migrate at all. These results align with the principles of electrophoresis, where charge and size influence the migration rate of molecules through a gel matrix.

In the DNA extraction experiment, we observed the successful extraction of DNA from wheat bacterium. The extracted DNA appeared as a thick web of strands on the spooling rod, resembling wet cotton. This confirmed the effectiveness of the DNA extraction procedure.

Discussion

The agarose gel electrophoresis results demonstrated the separation of dyes based on their charge and size. Negatively charged dyes migrated faster due to their attraction to the positive electrode, while positively charged dyes migrated more slowly. The neutral dye did not migrate, as it was not influenced by the electric field. These findings are consistent with the fundamental principles of electrophoresis.

In the DNA extraction experiment, the successful extraction of DNA from wheat bacterium was confirmed by the appearance of a thick web of strands on the spooling rod. This visual confirmation indicated that the DNA had attached to the rotating rod during the aqueous-alcohol interface. The extraction process was effective in isolating the DNA from cellular components.

Conclusion

In conclusion, this laboratory experiment provided valuable insights into agarose gel electrophoresis and DNA extraction techniques. Agarose gel electrophoresis allowed us to observe the separation of dyes based on their charge and size, in accordance with the principles of electrophoresis. The successful extraction of DNA from wheat bacterium was confirmed by the appearance of DNA strands on the spooling rod, demonstrating the effectiveness of the DNA extraction procedure.

Recommendations

Based on the results and observations of this experiment, the following recommendations can be made:

1. Continue exploring and experimenting with agarose gel electrophoresis to understand its applications in molecular biology.
2. Further investigate DNA extraction techniques and explore their potential applications in genetic research.

These recommendations can help expand our understanding of molecular biology and DNA technology.