Molecular Biology: A Practical Guide - Velikov V.A. 2013

Nucleic Acid Electrophoresis
Preparation of Agarose Gel

Electrophoresis is an electrokinetic phenomenon involving the movement of dispersed phase particles (colloidal solutions) in a liquid medium under METABOLISM/18.html">The Influence of an electric field. This phenomenon was first discovered in 1809 by Moscow University professors P.A. Strakhov and F.F. Reuss.

Agarose gel electrophoresis is a standard method for the Separation, identification, and purification of intact DNA molecules and their fragments (high-molecular-weight chromosomal DNA is invariably fragmented during isolation from Cells). Agarose is a fraction of the natural polysaccharide Agar.

DNA is a weak acid; therefore, it migrates toward the anode («+») due to its negatively charged phosphate groups. The movement of DNA (RNA) within the gel slab can be monitored because bands of fluorescent dye-stained DNA—formed by molecules of identical size migrating through the gel pores—are visible under UV light. Ethidium bromide (EtBr) is commonly used to stain DNA (λ_max = 590 nm). EtBr molecules intercalate into DNA, meaning they insert themselves between adjacent Base Pairs. The fluorescence intensity of bound EtBr is 20 times higher than that of free EtBr. This staining method provides high sensitivity, allowing as little as 10 ng of DNA to be visualized as an orange band. Other Dyes are also used, notably SYBR Green (λ_max = 497 nm). The wavelength of the UV transilluminator used for DNA visualization is 305-320 nm.

The rate at which DNA (RNA) migrates through agarose gel pores during electrophoresis is determined by molecular size and conformation. Linear double-stranded DNA molecules move through the gel matrix at speeds inversely proportional to the decimal logarithm of their molecular weights. Smaller fragments migrate ahead, while larger molecules move more slowly due to greater steric resistance. Among native nucleic acid molecules, tRNA (70-90 NUCLEOTIDES in size) and 5S rRNA (120 nucleotides) migrate the fastest. The largest genomic (chromosomal, nuclear) DNA fragments can reach sizes of up to 100,000 bp. To determine fragment sizes, molecular weight markers (DNA Ladders) are loaded into adjacent gel wells.

Gel casting. Agarose melts readily upon heating to 95 ^oC (in electrophoresis buffer). When the molten agarose is poured into a mold and allowed to set, it forms a transparent, resilient gel. Wells (pockets) for loading DNA into the gel are formed by placing a Teflon comb with Teeth into the unsolidified gel and removing it after the gel slab has polymerized.

Agarose concentration in the gel. The migration rate of DNA through gel pores depends on the agarose concentration. Knowing the sizes of the DNA molecules in a mixture, one can select the optimal concentration to achieve the best separation (Table 2).

Class="center">Table 2. Dependence of DNA fragment separation efficiency on agarose concentration in the gel

Staining DNA in agarose gels. To visualize DNA, ethidium bromide (EtBr; 3,8-diamino-5-ethyl-6-phenylphenanthridinium bromide) is used as a stain. Dye molecules intercalate (insert themselves) between adjacent DNA base pairs. Ethidium bromide is typically added to the molten agarose before gel polymerization. The absorption maximum of EtBr occurs at wavelengths of 300 and 360 nm, while emission takes place in the red-orange region of the visible spectrum at 590 nm.

Materials and Equipment

Agarose, comb, horizontal gel casting tray (often referred to as a «mold»; the lid of an immunological multiwell plate can also be used).

Solutions

- 5× TBE electrophoresis buffer. To prepare 1 L of buffer: Tris-OH (base) - 54 g; boric acid - 27.5 g; 0.5 M EDTA, pH 8.0 - 20 mL.

- EtBr. 10 mg/mL solution in Water. Store in the dark at 4 °C.

Procedure

1. Weigh out the calculated amount of agarose powder (1 g to prepare 100 mL of a 1% gel^a) and place it into a heat-resistant Glass beaker. Add 20 mL of 5× TBE electrophoresis buffer to the beaker and bring the volume to 100 mL with water.

2. Heat the mixture on a hot plate or in a microwave oven until completely melted.

3. Cool the molten agarose to 50-60 °C (the beaker should feel warm to the Touch). Add EtBr to a final concentration of 0.5 μg/mL: the technician should take 5 μL of the 10 mg/mL EtBr stock solution ^b per 100 mL of molten gel.

4. Position the comb approximately 1 cm from the edge of the tray. Pour the mixed molten agarose into the tray.

5. Once the gel has completely polymerized (30 min), carefully remove the comb by gently rocking it from side to side and pulling straight up.

Notes

^a Gel volume required for different tray types: an 11×11 cm tray requires approximately 110-140 mL, whereas an immunological plate lid requires about 30 mL.

^b Ethidium bromide is a potentially hazardous substance due to its ability to bind to DNA and promote DNA strand breaks when exposed to ultraviolet light. While definitive proof is lacking, certain hepatic enzyme oxidation products of EtBr exhibit slight yet measurable mutagenic activity. Always wear rubber gloves and follow standard safety precautions! Avoid Skin contact! EtBr degrades when exposed to light; therefore, gels should be stored in the dark at +4 °C for short periods only.