Agarose gel electrophoresis is primarily used in most research labs to either analyze the size of the DNA or to separate it for further purification. The separation of DNA fragments is on the basis of size.
Before understanding Agarose gel electrophoresis let’s look into some basic terms –
Agarose – It is a polysaccharide isolated from marine algae. Depending on the percentage of agarose, 50 nm to 200 nm web like channels or pores are formed that helps in the separation of DNA. Pores are like sieves that allow the movement of smaller molecules faster than larger molecules.
Polysaccharides – These are polymeric carbohydrates formed of small sugar molecules (Example - Glucose, Fructose etc.). These sugar molecules also referred to as monosaccharides are linked to each other by glycosidic bonds.
Electrophoresis – It is a technique widely used in research laboratories to separate biological macromolecules by inducing an electric current.
DNA – DNA or deoxyribonucleic acid stores all the genetic information that is passed from one generation to another (parent to child). This information includes features such as skin color, behavior, immunity and also specific species identity (i.e., Human). DNA is found in almost all cells of our body (there are always exceptions in biology) and is the same. It’s a polymer that is formed of codes (known as nucleotides) – Adenine (A), Thymine (T), Cytosine (C) and Guanine (G). In general, the information stored in DNA is essential for basic cellular physiology. These directly regulate all the events such as breathing, the beating of the heart etc.
Nucleotides – These are four codes that form the DNA (ATGC). It is made of a sugar moiety, attached with three phosphate molecules and a base. This base distinguishes the four codes and is formed of Carbon, Hydrogen, Oxygen and Nitrogen.
Ingredients required –
1. Agarose Gel
2. Fluorescent dye
STEP I – Make agarose gel
The percentage of agarose gel depends on the size of DNA fragment you need to separate or analyze. Smaller the DNA fragment, higher should be the percentage of agarose gel. Typically, 1% agarose gel is the standard, however, if the DNA fragment is smaller (0.2-1 kb), then 2% agarose gel is recommended. Similarly, for a DNA fragment of larger size (5-10 kb) 0.8% gel is usually used.
Agarose is mixed with TAE buffer. TAE is made of Tris-Acetic acid-EDTA. Tris is a strong base whereas acetic acid is a weak acid. Thus, the pH is 8-8.5, protecting the DNA from hydrolysis. EDTA chelates magnesium ions that act as a cofactor to DNase (DNA digesting enzyme). This further protects the DNA during the electrophoresis run.
Agarose forms gel with TAE upon heating. Latter when the temperature drops to approximately 50–60°C fluorescent dyes such as ETBR or SYBR green are added.
Further this gel is poured into the gel casting tray and a comb is attached for the loading wells to form.
Once the gel solidifies, remove the comb and place the gel with the casting tray on the electrophoresis tank. Always remember ‘Sample run towards red’ i.e., positive electrode, so place the gel accordingly.
Recipe for 50X TAE (one liter) -
Glacial Acetic Acid
STEP II – Mix sample with dye
The samples (to be separated or analyzed) are mixed with a fluorescent dye such as ethidium bromide or SYBR green. These dyes on binding with DNA start or increase their fluorescent intensities that can further be quantified under UV light.
STEP III – Load the sample and start the current
Load the samples using micropipettes in the wells. Always make sure to add a DNA ladder. It’s a kind of ruler that indicates the size of separated DNA fragments. It also acts as a positive control and will indicate whether the gel has run properly or not.
Start the electrophoresis power unit and check the dye front (usually a yellow or blue line). It is an indication of how much the samples have run. The higher the separation or resolution required the more the sample is to be run.
STEP IV – Visualization of DNA sample
Fluorescent dyes mixed with DNA samples illuminate under UV light. Thus, we can visualize our sample DNA under UV light through an instrument referred to as Gel Doc (Fig. 1). Lane 1 of figure 1 indicates the DNA ladder, lane 2 and lane 3 sample DNA.