Gel Electrophoresis is a technique used to separate DNA fragments or macromolecules like RNA and proteins, based on the sizes of the fragments. The shorter the fragment the faster it will move in the gel as it gets least resistance by the gel molecules. This technique is used to check whether the methods like PCR has worked properly or to check whether our plasmid has the right gene inserted.
Electrophoresis involves running a current through a gel loaded with the molecules of interest. The movement of the samples is directed based on the charge that the molecule carries. Based on the size and charge, the molecules will travel through the gel at different speeds, allowing them to be separated from one another. Since, all the DNA molecules possess same amount of charge per mass, the gel electrophoresis separates them on the basis of size only.
Making the buffers:
- Prepare a 50x stock solution of TAE buffer in 1000m of distilled H2O:
For this weigh 242 g of Tris base in a chemical balance. Transfer this to a 1000ml beaker.
Prepare EDTA solution (pH 8.0, 0.5M) by weighing 9.31g of EDTA and dissolve it in 40ml distilled water. EDTA is insoluble and it can be made soluble by adding sodium hydroxide pellets. Check the pH using pH meter. Make the solution 100ml by adding distilled water.
Pipette out 57.1 ml of glacial acetic acid.
Mix the Tris base, EDTA solution and glacial acetic acid and add distilled water to make the volume to 1000ml
- Prepare sufficient electrophoresis buffer (usually 1x TAE ) to fill the electrophoresis tank and to cast the gel:
For this we take 2ml of TAE stock solution in an Erlenmeyer flask and make the volume to 100ml by adding 98ml of distilled water. The 1x working solution is 40 mM Tris-acetate/1 mM EDTA
It is important to use the same batch of electrophoresis buffer in both the electrophoresis tank and the gel preparation.
- Prepare a solution of agarose in electrophoresis buffer at an appropriate concentration:
For this usually 2g of agarose is added to 100ml of electrophoresis buffer.
Agarose Concentration in Gel (% [w/v]) Range of Separation of Linear DNA Molecules (kb)
- Prepare bromophenol blue:
Gel loading dye helps us to track the movement of the DNA sample that is loaded in the gel wells. This mixture allows the DNA sample to sink into the gel wells and avoid the fragments to float. Gel loading dye is typically made at 6X concentration.
0.25% of bromophenol blue
0.25% of xylene cyanol
A Gel in the Gel Electrophoresis:
A gel in case of DNA separation is often made of a polysaccharide termed as agarose. Agarose is first mixed in a buffer (1X TAE Buffer) and heated to get it mixed and then allowed to cool down and Ethidium bromide(EtBr) usually a concentration of 0.5µg/mL, is then added which is a staining dye which illuminates under UV trans illuminator making DNA fragments visible. EtBr binds to hydrophobic interiors of the DNA fragments. Usually 1% gel is made for DNA fragments.
At the molecular level, the gel is a matrix of agarose molecules that are held together by hydrogen bonds and form tiny pores. When the agarose is heated in a buffer (water with some salts in it) and allowed to cool, it will form a solid, slightly squishy gel. At the molecular level, the gel is a matrix of agarose molecules that are held together by hydrogen bonds and form tiny pores.
At one end, the gel has pocket-like indentations called wells, which are where the DNA samples will be placed.
How do DNA fragments move through the gel?
The samples of DNA fragments are mixed with Bromophenol blue, which is a tracking dye and lets us see if the sample is moving. In the first well a DNA ladder is loaded, which acts a standard reference as it contains DNA fragments of known length. Commercial DNA ladders come in different size ranges, so we would want to pick one with good “coverage” of the size range of our expected fragments.
Now, the power to the gel box is switched on, and current begins to flow through the gel. The DNA molecules have a negative charge because of the phosphate groups in their sugar-phosphate backbone, so they start moving through the matrix of the gel towards the positive pole or anode. When the power is turned on and current is passing through the gel, the gel is said to be running. A typical voltage for running an agarose DNA gel would be in the range of 80-120V. A higher voltage will make the gel run faster, but may also melt it if it runs for a long period of time. A lower voltage will make the gel run more slowly.
As the gel runs, shorter pieces of DNA will travel through the pores of the gel matrix faster than longer ones as they gets the least resistance. After the gel has run for a while, the shortest pieces of DNA will be closest to the positive end of the gel, while the longest pieces of DNA will be found near the wells.
Observing Separated DNA fragments:
- When electrophoresis has completed, switch off the power supply and remove the lid of the gel electrophoresis box.
- Remove gel from the gel box. Drain off excess buffer from the surface of the gel. Place the gel tray on paper towels to absorb any extra running buffer.
- Remove the gel from the gel tray and expose the gel to uv light, place the gel in the UV trans illuminator. DNA bands should show up as orange fluorescent bands.
- Properly dispose of the gel and running buffer per institution regulations. Boil the gel to degrade EtBr.