MOLECULAR BIOLOGY AND GENETIC ENGINEERING

Experiment 3

INTRODUCTION:

Agarose gel electrophoresis is a method used to separate DNA and RNA molecules according to their molecular size. This is achieved when negatively charged nucleic acids migrate through an agarose gel matrix under the influence of an electric field (electrophoresis). Shorter molecules move faster and migrate farther than the larger ones. This method is simple, rapid to perform, and capable of resolving fragments of DNA that cannot be separated by other procedures such as density gradient centrifugation. The position of DNA in the agarose gel is visualized by staining with low concentration of fluorescent intercalating dyes, such as Ethidium bromide.

Principle

Agarose is an unmodified polysaccharide of galactose with neutral charge, which is essential to prevent interactions with charged DNA and protein molecules. It forms large pores which is useful for separation of DNA and proteins by its molecular size. In aqueous solution, below 35oC these polymer strands are held together in a porous gel structure by non-covalent interactions like hydrogen bonds and electrostatic interactions. On heating the solution, these non-covalent interactions are broken down and the strands are separated. As the solution cools, these non-covalent interactions are re-established and the gel is formed. Purified agarose is insoluble in water or buffer at room temperature but dissolves on boiling. As it cools, agarose undergoes polymerization i.e., sugar polymers cross-link with each other and cause the solution to solidify, the density or pore size of which is determined by concentration of agarose.

• The following factors determine the rate of migration of DNA through agarose gels:

1. Molecular size of DNA: Fragments of linear DNA migrate through agarose gels with a mobility that is inversely proportional to the log10 of their molecular weight. Larger molecules migrate more slowly because of greater frictional drag as they pass through the pores of the gel less efficiently than the smaller molecules. Circular forms of DNA migrate in agarose differently from linear DNA of the same mass. Undigested plasmids migrate more rapidly than the same plasmid when linearized.

2. Agarose Concentration: By using gels with different concentrations of agarose, DNA fragments of different sizes can be resolved. Higher concentration of agarose facilitates separation of small DNA, while lower agarose concentration allows resolution of larger DNA.

3. Electrophoresis buffer: Several buffers have been recommended for electrophoresis of DNA. The most commonly used are TAE (Tris-acetate-EDTA) and TBE (Tris-borate-EDTA). DNA fragments will migrate at different rates in these two buffers due to difference in ionic strength. Buffers not only establish a pH, but provide ions to support conductivity.

4. Effect of Ethidium bromide: Ethidium bromide is a fluorescent dye which fluoresces at 254- 366nm. It intercalates between bases of nucleic acids and allows very convenient detection of DNA fragments in gels on a UV transilluminator. Binding of ethidium bromide to DNA alters its mass and rigidity, and therefore its mobility.

5. Voltage: As the voltage applied to a gel is increased, larger fragments migrate proportionally faster than smaller fragments. The best resolution of fragments larger than about 2 kb is attained by applying not more than 5 volts per cm to the gel (the cm value is the distance between the two electrodes, and not the length of the gel).

Materials required

Glasswares: Conical flask, Measuring cylinder, Beaker

Reagents: Distilled water, Ethidium bromide (10 mg/ml), Genomic DNA, Plasmid DNA, Agarose, 50X TAE, 1 kb DNA Ladder and 6X Gel Loading Buffer.

Other requirements: Electrophoresis apparatus, UV Transilluminator, Micropipettes, Tips, Adhesive tape, Microwave/Burner/Hotplate.

Procedure

1. Preparation of 1X TAE (gel electrophoresis buffer): To prepare 500 mL of 1X TAE buffer, add 10 mL of 50X TAE Buffer (Product Code: ML016) to 490 mL of sterile distilled water*. Mix well before use. * Molecular biology grade water is recommended (Product code: ML024).

2. Prepare gel tray by sealing the ends with adhesive tape. Place comb in the space provided on the gel tray.

3. To prepare 50 mL of 1 % agarose solution, add 0.5 g agarose (pre-weighed [DS0237], provided) to 50 mL 1X TAE buffer in a glass beaker or flask. Heat the mixture on a microwave/burner/hot plate, swirling the glass beaker/ flask occasionally, until agarose dissolves completely (Ensure that the lid of the flask is loose to avoid buildup of pressure).

4. Allow solution to cool down to about 55-60°C. Add 0.5 µL Ethidium bromide (10mg/ml), mix well and pour the gel solution into the gel tray. Allow the gel to solidify for about 30 minutes at room temperature.

5. To start the run, carefully remove the adhesive tape from both the ends of the gel tray, place the tray in electrophoresis chamber, and fill the chamber (just until wells are submerged) with 1X TAE electrophoresis buffer and gently remove the comb.

6. To prepare samples for electrophoresis, add 2 µL of 6X gel loading buffer (Product code: ML015) for every 10 µL of DNA sample. Mix well and load the sample into the well. One can also load 3 µl of 100 bp ladder (Product code: MBT049; MBT130) or 1kb DNA Ladder (Product code: MBT051) into one well.

7. Connect the power cord to the electrophoretic power supply according to the conventions: RedAnode and Black- Cathode. Electrophorese at 100-120 volts and 90 mA until dye markers have migrated an appropriate distance, depending on the size of DNA to be visualized.

8. Electrophoresis apparatus should always be covered with the lid to avoid electric shocks. Avoid use of very high voltage as it can cause trailing and smearing of DNA bands in the gel, particularly with high molecular weight DNA.

9. Switch off the power supply once the tracking dye from the wells reaches 3/4th of the gel which takes approximately 45 minutes.

Precautions:

Safety Precaution: UV light can damage the eyes and skin. Always wear suitable eye and face protection when working with a UV light source. UV light damages DNA. If DNA fragments are to be extracted from the gel, use a lower intensity UV source if possible and minimize exposure of the DNA to the UV light.

Disposal of Ethidium bromide waste: All items that were in contact with Ethidium bromide must be disposed off in the designated waste container (marked with "Ethidium bromide waste") within the Gel-Doc-Area, including gels, tissue paper to clean UV table, and nitrile gloves.

Hazard: Ethidium bromide is a powerful mutagen and is very toxic. Appropriate safety precautions should be taken by wearing latex gloves; however, use of nitrile gloves is recommended.

Observation and Result:

The illumination of a stained gel under UV light (254–366 nm) allows DNA bands to be visualized against a background of unbound dye. The gel image can be recorded by taking a Polaroid™ photograph or using a gel documentation system.

Interpretation:

1. Agarose gel electrophoresis and spectrophotometric analysis will confirm the yield and purity of DNA.

2. Genomic DNA being of larger molecular size migrates at a lower speed than the plasmid DNA of smaller molecular size.

3. If RNA contamination is present, one would see a faint and smeary RNA band below the genomic and plasmid DNA since RNA being of lower molecular weight runs faster than the genomic and plasmid DNA.

References:

1. https://himedialabs.com/TD/HTBM001.pdf

2. T.A Brown - Gene cloning.

3. Wilson and Walker- Principles and Techniques of Biochemistry and Molecular Biology.

Questions

1. What if there light or no band observed on gel? What are the possible reasons and the solution for the same?

2. What is the possible reason for the smeared band formation and justify the solution for the same?

3. What are the possible reasons for the improper gel solidification? Also mention the solution.