The Use of Gel Electrophoresis - Lab Report Example

DNA is a double-stranded nucleic acid and both the strands run antiparallel. It is made up of pentose sugar (de-oxy ribose), phosphate group, and nitrogen base pairs (Double ringed Purines: Adenine and Guanine and Single ringed Pyrimidine: Cytosine and Thymine). One purine pair with one Pyrimidine with hydrogen bond to make the double-stranded DNA. Adenine (A) pairs with Thymine (T) with double H-bond and Guanine (G) pairs with Cytosine (C) through triple H-bond.

The isolation procedure requires the disruption of cells so that the cell content comes out, followed by sedimentation of the cellular debris on the application of centrifugal force and to collect the DNA from the supernatant. These DNA fragments are separated using gel electrophoresis. The process encompasses separation, based on their size, the pore size of the gel, the voltage gradient applied, and the salt concentration of the buffer. Larger pore size is for the separation of fragments larger than 500- 1000 bp and smaller pores of agarose gels are used to resolve fragments smaller than 1000 bps and can be visualized. The technique of electrophoresis is based on the fact that since DNA contains a phosphate group, it is negatively charged at the neutral pH. When an electric potential is applied, it moves toward the positive terminal.

The solidified agarose gel is inserted into the electrophoresis chamber and is just covered with buffer. The DNA sample is mixed with the loading buffer and then pipette in the sample wells. On application of the current DNA migrates toward the positive (red-colored) electrode. The distance DNA has migrated in the gel can be judged by visually monitoring the migration of the tracking dyes. After adequate migration, DNA fragments are visualized by ethidium bromide. This is a fluorescent dye and it intercalates between the bases of DNA and RNA. It is incorporated into the gel so that staining occurs during electrophoresis. Bands appear on the gel and can be visualized.

Results:

Part 1

A fluffy white layer was formed at the boundary between the green and the purple liquids when the ethanol was added. It was made up of fine filaments.

Part 2

After putting the electrical current, strands of the DNA become visible to the naked eye. It becomes like stains, or bands, on the gel.  

Discussion

NaCl removes protein and carbohydrate in DNA and also act as a lysing buffer. NaCl contains Na+ which binds with the negatively charged phosphate molecule of the DNA. It also stabilizes the pH and processes the density of DNA. Washing liquid reduces the acidity of the solution and removes CO2.

Detergents remove the interfering cells and are used as a substitute for the chemical compound that is capable of damaging the cell wall and membrane. They act as emulsifying agents and can digest compound that causes the stiffness of polymeric cells.

Endiamin tetra ethyl acetate (EDTA) serves to remove the Mg+2 ion and proven enzymes that can damage cellular DNA, it protects the DNA from DNAse. It interrupts the interaction of the polar cell membrane and unites as a detergent.

Gel electrophoresis is a powerful tool for the separation of macromolecules with different sizes and charges. DNA molecules have an essentially constant charge per unit mass thus they separate in agarose, based on the size, the smaller the size more distance it can travel, and the larger the size of the DNA less it can travel. Increasing the concentration of a gel reduces the migration speed and enables the separation of smaller DNA molecules. The higher the voltage, the faster the DNA moves but higher voltage can cause the gel to melt.

A fluffy white layer was formed between the green and purple layers because of the emulsification of the organic cellular molecules with the washing liquid.

If the cleaning process is not performed with precision and care is not taken then a distinct band with a diffuse staining region just ahead or behind the main band appears on the gel. The reason for this is either sample degradation or partially denatured DNA. These are fuzzy bands and indicate the lack of precision maintained in the procedure of isolation.

Conclusion

Centrifuges separate substances based on molecular weight, therefore substances lighter in weight will be on the top. To isolate the DNA, membranes and cell wall are removed. For this tissues are isolated first with the help of salt solution and washing up liquid. As higher temperature increases the permeability of the cell membrane the temperature is set at 60° C. Tissues of kiwi fruit when mixed with salt and washing up liquid, enable the disruption of the cell wall and the cell membrane, and the cellular contents come out. Upon centrifugation, the cell debris settles at the bottom and the supernatant contains light molecular particles including the DNA. To get the purified DNA which is devoid of other protein molecules and histones, ethanol treatment is given. Ethanol cleans the DNA from the cellular material including small organic molecules.

The protocol for DNA isolation and visualization works at the rate of 100% if appropriate care and precision are performed, right from the preparations of solutions and maintaining the appropriate temperature of the solutions and reagents.