Category: Gel electrophoresis

Migration of Bromophenol Blue and Xylene Cyanol in Agarose Gel Running in TBE or TAE Electrophoresis Buffer

Bromophenol blue and Xylene cyanol are the two most commonly used tracking dyes for the analysis of DNA on agarose gel electrophoresis. These negatively charged dyes not only help in monitoring the progress of agarose gel electrophoresis but also allow easy monitoring of sample loading process onto the wells of agarose gel. Their position in relation to DNA fragments is an important information that helps to determine how far a gel must be run without accidentally letting the DNA fragments of interest run out of the gel and at the same time ensuring good resolution among different size DNA fragments.

Detection of DNA in Agarose Gel

♦ After the gel electrophoresis has been completed, the next step is to analyze agarose gel for DNA fragments. Unfortunately,…

Nucleic Acid Staining Dyes for Detection of DNA in Agarose Gel

There are many nucleic acid staining dyes that can be used to visualize DNA embedded in the agarose gel. These…

Preparation of 50X TAE Electrophoresis Buffer

A 50x TAE buffer can be prepared by mixing and dissolving 242 g Tris base, 100 ml of 0.5 M EDTA and 57.1 ml glacial acetic acid in a deionized water to a final volume of 1000 ml. The pH of the final solution should be between 8.2 – 8.4. 

Preparation of 5X TBE Electrophoresis Buffer

TBE is a very common electrophoresis buffer for DNA agarose gel electrophoresis. It is prepared as a 5X stock solution that can be diluted to 1x or 0.5x working solution for DNA agarose gel electrophoresis. The 5X concentrated solution contains 0.445 M Tris borate and 0.01 M EDTA (pH 8.2 – 8.4).

A 5X stock solution is prepared by dissolving 54 g Tris base, 27.5 g Boric acid, and 20 ml of 0.5 M EDTA in water to a final volume of 1000 ml. The solution is sterilized by autoclaving and is stored at room temperature.

Agarose Gel

Agarose gel can be used for the separation of nucleic acids (DNA and RNA) and high molecular weight proteins or protein complexes.

Agarose

Agarose is a useful matrix for a number of analytical and preparative techniques including gel electrophoresis, chromatography, and support matrix to immobilize enzymes and cells. Agarose is available in a variety of forms, which differ in physical properties. The two most common agarose are standard agarose and low melting agarose. Standard agarose is most commonly used for analysis of DNA and RNA. Low melting agarose is often used for preparative purposes such as elution of DNA fragments and complexes.

Protocol: Running DNA Samples in Agarose Gel

Agarose gel is placed in an electrophoresis tank filled with an electrophoresis buffer. DNA samples are mixed with DNA loading dye and loaded onto the wells of agarose gel. Electrophoresis apparatus is connected to electric supply and electrophoresis is performed at constant voltage until the desired separation among DNA fragments is achieved. After the run is over, gel is analyzed using UV-transilluminator or Gel Doc system.

Agarose Gel Preparation Process

Protocol: Preparation of Agarose Gel for DNA Analysis

Agarose Gel is a gelatin-like slab, which contains small wells for loading DNA samples. It is prepared by melting agarose in a suitable electrophoresis buffer. Molten agarose is then poured into a specialized tray (casting tray), which controls the size and shape of the gel. The comb is used to create wells in agarose gel.

DNA Loading Dye

DNA sample is mixed with DNA loading dye (also called sample loading dye) prior to loading onto the wells of agarose gel for electrophoresis. DNA loading dyes contain a high-density reagent and tracking dyes. DNA loading dye makes the DNA sample heavier and coloured, thus helping the DNA sample to sink into the agarose wells without diffusing out and enabling us to monitor the loading process. In addition, tracking dyes in loading dye migrate as a diffuse band to the same direction as DNA which can be seen visually thus allowing us to monitor the progression of electrophoresis. However, tracking dyes sometimes interfere in the analysis of DNA bands by masking it.