Agarose gel electrophoresis

BACKGROUND OF AGAROSE GEL ELECTROPHORESIS

Agarose gel electrophoresis is a method of gel electrophoresis used in biochemistry, molecular biology, genetics, and clinical chemistry to separate a mixed population of macromolecules such as DNA or proteins in a matrix of agarose, one of the two main components of agar.

Aim of the experiment is to perform the agarose gel electrophoresis of a isolated genomic DNA sample.

When an electric field is applied across the gel DNA, which is negatively charged at neutral pH, migrates towards the anode. The rate of migration is determined by a number of parameters like molecular size of DNA, agarose concentration, conformation of the DNA, applied voltage, presence of intercalating dyes and composition of electrophoresis buffer.^1,2 

Molecules of double stranded DNA migrate through the gel matrices at the rates of that are inversely proportional to the log₁₀ of the number of base. Thus, the larger molecules migrate slowly due to greater frictional drag than the smaller molecules. Genomic DNA being heavier usually remains just below the well.¹ 

In brief, agarose gel electrophoresis is normally used to separate DNA (single-stranded, double-stranded, and supercoiled) and RNA. Since DNA is negatively charged, it migrates in an electric field toward the positively charged cathode.

The agarose matrix retards DNA migration roughly proportionally to DNA length when the DNA being separated is small. Longer oligonucleotides have a harder time traveling through the matrix, while shorter oligonucleotides (and small molecules such as ATP) breeze right through it. The general equation for the relationship between electrophoretic mobility (µ) and the gel concentration (C) is:² 

log µ = log µ_o – K_r C

where, µ = electrophoretic mobility of DNA

µo = free electrophoretic mobility of DNA

K_r = retardation coefficient

C = concentration of gel

Agarose Gel Concentration (%w/v)	DNA Size Range for Optimal Separation
0.3	5,000 – 60,000
0.6	1,000 – 20,000
0.7	800 – 10,000
0.9	500 – 7,000
1.2	400 – 6,000
1.5	200 – 3,000
2.0	100 – 2,000

Different concentration of agarose is used for different size of DNA molecules.³

Ethidium bromide, a fluorescent dye that binds to DNA molecule by intercalating between adjacent base pairs. It causes partial unwinding of the double helix. The DNA-ethidium bromide complex strongly adsorbs UV light at 300nm, retains some energy and reemits visible light at 590 nm.

REQUIREMENTS

Buffers and solutions (reagents):

Agarose solutions

Ethidium Bromide – 10 mg/ml

Dissolve 100 mg of EtBr in 10 ml of double distilled water (DDW). Stir on a magnetic stirrer until the dye has dissolved. Dispense into 500 µl aliquots and wrap the container in aluminium foil and store at room temperature. Final concentration is 0.5 µg/ml.

Ethyl alcohol (75%)

Electrophoresis buffer^4,5

50X TAE: Take the components i.e., 24.2 g Tris base, 5.71 ml glacial acetic acid and 10 ml of 0.5 M EDTA (pH 8.0) and make up the volume 100 ml with double distilled water. Sterilize by autoclaving and store at room temperature.

IX TAE: take 10 ml of 50X TAE in 490 ml of DDW.

Gel loading dye (6X): Take the components of 0.25% Bromophenol blue, 0.25% Xylene cynol in 30% glycerol and store at 4^°C as 1ml aliquots.

Nucleic Acids and oligonucleotides:³

DNA samples

DNA Ladders

Equipments

Horizontal electrophoresis system

Power pack

Autopipettes (1 ml, 200 µl, 10 µl)

Tips (1 ml, 200 µl, 10 µl)

Cellophane tape

PROCEDURE

Firstly, clean the gel tray and the comb with 75% alcohol carefully. Then, seal the open ends of the clean plate by the cellophane tape and keep on the bench. After completing the initial steps, take 20 ml of IX TAE in an Erlenmeyer flask and add 2 g agarose to it.

The liquid is the kept inside the micro-oven until a clear, transparent solution is achieved. When the solution cools down to 60^°C then add 10 µl of EtBr to obtain a final concentration of 0.5 µg/ml and mix thoroughly. The comb has to be placed at a distance of 0.5-1.0 mm above the tray to get a complete well.

The wells are then filled with the warm agarose solution (15 ml) by pouring into the tray and allow to become hard which forms a matrix. Remove the comb carefully after the gel is completely set (30-40 min at RT) and transfer the gel to the electrophoresis tank containing IX TAE electrophoresis buffer.

Then the sample (DNA) has to be prepared with the gel loading dye and buffer (gel loading dye 1.5µl, IX TAE 5.5 µl and isolated sample DNA 2.0 µl) and slowly load into the slots of the submerged gel electrophoresed at 80 volt and 100mA current. After keeping it for 45 min visualize the gel in an UV transilluminator.²

Precaution³

1. Same batch of electrophoresis should be used in both the electrophoresis tank and the gel.
2. Stock solution of ethidium bromide should be stored in light tight containers (e.g, brown bottle or a bottle completely wrapped in a aluminium foil) at room temperature.
3. After pouring the warmed agarose, the air bubbles should be removed under or between the teeth of the comb, if any.

Care should be taken at the time of loading , so that the sample will not spill-out from the well.

CONCLUSION

From the above experiment it can be concluded that DNA having higher molecular weight cannot travel higher distances so they separate out from that of lower molecular weight of DNA which can be seen in the lower subsequent bands. Thus, the length of the nucleotide can be determined.

REFERENCES

1. Kryndushkin DS, Alexandrov IM, Ter-Avanesyan MD, kushnirov VV(2003).
2. Sambrook j, Russel DW(2001). Molecular cloning: A laboratory Manual 3^rd Ed. Cold spring harbor lab press.
3. Donald Voet; Judith G. voet(1995) biochemistry(2^nd ed.)
4. John wiley & sons. pp 877-878. ISBN 978-0471586517
5. DNA revealed (PDF). National centre for biotechnology education. University of reading

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