Gel Electrophoresis: Lab Report
Introduction.
Electrophoresis is a technique used to separate and purify macro-molecules, especially proteins and nucleic acids that differ in size, charge or conformation. In this technique the sample to be tested is exposed to electric current and allowed to separate as the positive components of the sample are attracted towards the negative side and the negative towards the positive side. Proteins can have either a net positive or net negative charge, nucleic acids have a consistent negative charge imparted by their phosphate backbone, and migrate toward the anode. The movement of charged molecules is called migration. Molecules migrate towards the opposite charge. A molecule with a negative charge will therefore be pulled towards the positive end. (Tooze and Branden 1999).
In Gel Electrophoresis Proteins and nucleic acids are electrophoresed inside a matrix or "gel". The gel is cast in the shape of a thin slab, with wells for loading the sample. The gel is immersed within an electrophoresis buffer that provides ions to carry a current and the running buffer to maintain the pH at a relatively constant value. The gels that can be use are Agarose and Polyacrylamide depending on the specification of the sample as well as procedure. The gel consists of a permeable matrix “sieve” like, through which molecules can travel when an electric current is passed across it. Smaller molecules migrate through the gel more quickly and therefore travel further than larger fragments that migrate more slowly and therefore will travel a shorter distance. As a result, the molecules are separated by size (Wilson and Walker1986).
Agarose is a polysaccharide that is extracted from seaweed. It can be used in varying concentrations of 0.5 to 3%. The higher the agarose concentration, the denser the matrix and vice versa. Small fragments of DNA are separated on higher concentrations of agarose whereas larger molecules require a lower concentration of agarose. Agarose gels have a large range of separation, nevertheless they have relatively low resolving power. By varying the concentration of agarose, fragments of DNA from about 200 to 50,000 bp can be separated using standard electrophoretic techniques (Rao ,et al 1951).
Polyacrylamide is a cross-linked polymer of acrylamide. The length of the polymer chains is affected by the concentration of acrylamide used. Oxygen inhibits the polymerization process hence they must be poured between glass plates. Polyacrylamide gels have a small range of separation, however they have very high resolving power. In the case of DNA, polyacrylamide is used for separating fragments of less than about 500 bp. However, under appropriate conditions, fragments of DNA differing in length by a single base pair are easily resolved. In contrast to agarose, polyacrylamide gels are used for separating and characterizing mixtures of proteins (Wilson and Walker1986).
Materials and methods
Refer to practical schedule practical 5: DNA analysis by restriction enzyme digestion.
Alteration: no restriction enzymes were used. Expected are just single bands and not laddered bands.
Aim: To determine the movement and separation of plasmid DNA in an agarose gel electrophoresis.
Objectives:
Determine the migration speed of the components of the DNA samples used.
Compare movement of DNA of cabbage and plasmid DNA in a gel.
Understand the concept of how charge and molecular weight can be used to separate molecules using gel electrophoresis.
Establish the importance of factors affecting this technique
Results of this Lab report were removed purposely. Donate and access full content
Discussion
The experiment was not very successful this could have been caused by a number of factors which including magnitude of the charge, charge density, the molecular weight, the shape, the solution of pH, and temperature of the whole system. These could have affected the rate of migration of the DNA samples as well as their ability to move through the gel.
The ladder DNA was not smeared and as a result it was not visible on the gel.
Cabbage DNA from 2-4 were not clearly visible meaning that the DNA was not successfully broken down.
Sample 5 had a slightly visible smear hence DNA was broken down successful.
Sample in well 7 had a visible DNA smear indicating that DNA was successfully separate and broken down.
Samples in well 8-9 no DNA so there was no visible smear.
Sample in well 10 was to a lesser extent successfully broken down as indicted by the slightly visible smear.
Poor smearing and absence of other sample smears indicate that the samples place in the wells did not have concentration of the DNA hence they were not clearly visible.
Ethidium bromide is a fluorescent dye and it intercalates between nucleic acids bases and provides opportunity to easily detect nucleic acid fragments in gels. The gel subsequently was illuminated with an ultraviolet lamp which was placed on a light box the stained nucleic acid fluoresces reddish-orange. Migration of fragments in an agarose gel depends on the difference in electric current. Different optimal voltages are required for different fragment sizes (Lehniger 1984)
To improve resolution in the experiment the following could have been done:
apply higher voltage
increase ionic strength of the electrophoretic medium.
provide the whole system with an efficient cooling
Circular forms of DNA migrate in agarose distinctly differently from linear DNAs of the same mass. Uncut plasmids appeared to migrate more rapidly than the same plasmid when linearized.
Fragments of linear DNA migrated through agarose gel with a mobility that is inversely proportional to the log10 of their molecular weight. Several factors have important effects on the mobility of DNA fragments in agarose gels, and can be used in optimizing separation of DNA fragments. Among these factors are: Agarose Concentration, Voltage, Electrophoresis Buffer and Effects of Ethidium Bromide (Thermofisher 2015): (Millar 1998).
References
Lehniger. A.L. (1984) Principles of Biochemistry. Worth publishers Inc. New York
Millar. J. M (1998) Chromatography; concepts. Library of congress. Canada
Rao ,et al. (1951) Analysis of sugars using paper chromatography. Forest research Institute. Dehra Dun.
Shimadzu. R. (2016) Analysis of protein. All time publishers. Sweden
Tooze. J, Branden. C. (1999). Introduction to protein structure. Garland publication. New york
Thermofisher.(2015) Chromatographic Techniques. Cambridge University press, Great Britain.
Wilson , Walker. J. (1986) Principles and techniques of practical biochemistry, 4thedition, Cambridge University press, Great Britain.
