Preparation of Agarose gel

The materials or equipments that are used in the preparation of Agarose gel are simple and they include:

·   A chamber that is used for electrophoresis and power supply.

·   Trays for gel casting, these trays are available in a number of sizes and the plastic that is used is composed of UV. The ends of tray that are open can be closed with the help of tape and the completion of process the gel is removed.

·   Sample combs are also used and in order to form wells the molten medium is poured.

·   Buffer of electrophoresis is also used and the most common buffer is EDTA.

·   Another loading buffer is also used in this process, and this buffer consists of some dense material like glycerol. This buffer is used to pour the sample into the wells that are created by sample combs. This can also be used to estimate the speed of electrophoresis process.

·   Staining of the molecules is performed in order to make these molecules visible. In order to visualize DNA molecules we can use UV light and ethidium bromide can also be used for this purpose. When the molecules are separated by this process than they are stained. The gel can be soaked in the ethidium bromide solution. Ethidium bromide is not commonly used as it is a hazardous material and it can cause cancer.

·   Transilluminator is a box of UV light and it is used to observe the molecules which we have stained. Our eyes can be damaged by this process as UV light is used and protective measures are used. In order to prepare gel, we mix the powder of Agarose with the buffer of electrophoresis and make the concentration up to a desirable limit. Heat this mixture in oven. After heating add ethidium bromide in order to make the DNA visible. After this cool the solution and cooling temperature should be 60C and after that this solution is added into the tray and sample combs are present in the solution to create wells and solution is solidified.

·   When the gel is solidified than remove the combs. After that the gel is placed in the chamber used for electrophoresis and buffer is used to cover this. In the wells the loading buffer is added that will move the sample into the wells. After that current is supplied. The supply of current is checked by observing the bubbles that will produce from the electrodes. As DNA has negative charge than it will move towards the positive pole and the color shown by DNA is red.

·   Different dyes can be used to observe the distance that is being travelled by the DNA.

 

Preparation of polyacrylamide gel:

The materials and equipments that are used in the preparation of polyacrylamide gel are:

·   The acrylamide can be regarded as a neurotoxin and it can cause cancer so while dealing with polyacrylamide avoid the contact of skin with it. The protective measures include wearing of gloves during the preparation of polyacrylamide gel.

·   Use the chemical fume hood while dealing with silane as it is regarded as toxic.

·   In order to prevent the sticking of gel we can use glass plate treated with Gel Slick and a small glass plate is also used. These plates may be placed away from each other as they can cause cross contamination.

·   After the completion of process these plates are placed in NAOH solution in order to remove the materials with which they have been treated. After that also wash the plates with water and detergent.

·   In order to store gel we can use paper towel and also place the plastic wrap so that the gel may be prevented from drying.

Loading of sample:

·   In order to load gel the samples are denatured. If the annealing conditions are provided to DNA it can join again and can form fragments.

·   If the composition of gel is 6% than the migration of xylene cyanol will be 105 bases and that of bromophenol blue is 25 bases.

Staining of gel:

In order to stain the molecules we use different agents like for the staining of protein we can use coomasie blue. Other dyes which can be used for same purpose are amido black and ponceau red. We mostly use ponceau red as it can be easily removed from protein and protein can be analyzed.

Silver staining is another technique that can be used. For proteins mostly silver staining is used. For this process we dip the protein in the solution of silver nitrate. The metallic silver is precipitated.

·         The steps in which we deal with formaldehyde are necessary to perform in fume hood.

·         The solution is cooled at the temperature of 4C.

·         Must avoid from the silver staining at the start of the process.

·         Rinse the solution with water for 10 minutes and this time range should not be exceeded.

Although ethidium bromide and many other agents can be used to observe the molecules on the gel we can also use silver for the staining purpose as the ethidium bromide is a carcinogen. There is another possibility of autoradiogram which is performed when the DNA sequences contain radioactivity.

Migration of the fragments of DNA:

The DNA molecules move with the speed that is proportional to their molecular weight. The distance of DNA is observed from the wells towards the electrode as they move towards the positive pole. A straight line appears that show the distance of the DNA molecule. The circular forms of DNA are also present and their movement is different from the movement of linear molecules of DNA.

The plasmids that are not cut they can move with the greater speed. The uncut plasmids contain two different forms of DNA molecules [Brodjy and Kern. 2004]. There are many factors that affect the movement of DNA molecules in the gel.

These factors are:

Concentration of Agarose:

In order to separate out DNA molecules that are of different sizes we can use different concentrations of Agarose. If the concentration of Agarose gel is high than molecules of small size can be separated and if the concentration of Agarose gel is less than molecules of large size are separated.

Voltage:

As the voltage to applied to the matrix of gel by power supply than molecules move towards the poles. If the voltage applied is high than the molecules of large size will move with greater speed and if the voltage applied is low than the molecules of small size will move faster. If the fragment size is greater than 2 kb than the voltage applied is of 5 volts.

 

 

Buffer for electrophoresis:

Many buffers are recommended for use in electrophoresis process. The buffer that is mostly used is EDTA and TAE. As the ionic strengths of these buffers are different than the movement rate of DNA molecules will be different. Not only ions are supplied by buffer they can also maintain ph. If you had used water instead of using buffer than the movement of DNA molecules will be zero. By the use of buffer heat can also be generated.

Ethidium bromide:

Ethidium bromide is basically a dye that is used for the staining of nucleic acids and we can easily detect the molecules with the help of it. The gels are dipped in ethidium bromide either before the loading of sample or after the sample is being loaded. There is a risk that ethidium bromide if binds with DNA it can affect its movement and mass and also ethidium bromide is a carcinogen and can cause cancer.

Coomasie blue:

The reagents that are required for this type of staining are:

·         Distilled water.

·         Brilliant blue.

First of all we have to fix the solution and this fixing of solution requires different ratios of the materials like acetic acid, methane and water. The ratios of these agents are 10, 50 and 40 respectively.

The next step is to destain the solution. This step also involves the mixing of different agents in different ratios. The different ratios of acetic acid, methanol and water are used. These concentrations are 10, 45 and 45.

In the next step we concentrate the solution. In order to concentrate the solution we use 60 ml of methanol and 12g of BBR. Stirring of the solution is necessary after adding different agents.

Next step is to make working solution. This involves 30 ml of coomasie solution and 500ml of methanol. Another agent acetic acid is added in the concentration of 100ml and water is added in the concentration of 400 ml.

Silver staining:

If we want to detect very small concentrations of proteins like in the range of 1ng to 1mg than we use silver staining as this is very sensitive.

In case of coomasie solution if stain is removed than we cannot again stain but in case of silver staining if stain is removed than we can stain again.

Protocols:

If we want to destain which means that we want to remove the stain. This step is performed when all the bands become invisible. For this purpose we wash the gel with water for 10 min and this process is repeated for 3 to 5 times.