ABSTRACT
Electrophoresis may be a technique that permits separation and analysis of charged molecules in an electrical field. Gel electrophoresis is most ordinarily used for separation and purification of proteins and nucleic acids that differ in size, charge, or conformation. The gel consists of polyacrylamide or agarose. Agarose is acceptable for separating DNA fragments ranging in size from a couple of hundred base pairs to about 20 kb. Polyacrylamide is preferred for proteins and smaller DNA fragments. The mobility of DNA is constant under defined electrophoretic conditions. These conditions are characterized by the electrical parameters (current and voltage) and factors like buffer composition, agarose concentration, and temperature.
INTRODUCTION
Electrophoresis may be a technique won’t to separate and purify macro-molecules, especially proteins and nucleic acids that differ in size, charge or conformation. During this technique the sample to be tested is exposed to current and allowed to separate because the positive components of the sample are attracted towards the negative side and therefore the negative towards the positive side. Proteins can have either a net positive or net charge, nucleic acids have a uniform charge imparted by their phosphate backbone, and migrate toward the anode. The movement of charged molecules is named migration. Molecules migrate towards the other charge. A molecule with a 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 within the shape of a skinny slab, with wells for loading the sample. The gel is immersed within an electrophoresis buffer that gives ions to hold a current and therefore the running buffer to take care of the pH at a comparatively constant value. The gels which will be use are Agarose and Polyacrylamide counting on the specification of the sample also as procedure. The gel consists of a permeable matrix “sieve” like, through which molecules can travel when an electrical current is passed across it.
Smaller molecules migrate through the gel more quickly and thus travel further than larger fragments that migrate more slowly and thus will travel a shorter distance. As a result, the molecules are separated by size (Wilson and Walker1986).
Agarose may be a polysaccharide that's extracted from seaweed. It are often utilized in varying concentrations of 0.5 to 3%. The upper the agarose concentration, the denser the matrix and the other way around. Small fragments of DNA are separated on higher concentrations of agarose whereas larger molecules require a lower concentration of agarose. Agarose gels have an outsized range of separation, nevertheless they need relatively low resolution. By varying the concentration of agarose, fragments of DNA from about 200 to 50,000 bp are often separated using standard electrophoretic techniques (Rao, et al 1951).
Polyacrylamide may be a cross-linked polymer of acrylamide. The length of the polymer chains is suffering from the concentration of acrylamide used. Oxygen inhibits the polymerization process hence they need to be poured between glass plates. Polyacrylamide gels have alittle range of separation, however they need very high resolution. Within the case of DNA, polyacrylamide is employed for separating fragments of but about 500 bp. However, under appropriate conditions, fragments of DNA differing long by one nucleotide are easily resolved. In contrast to agarose, polyacrylamide gels are used for separating and characterizing mixtures of proteins (Wilson and Walker1986).
MATERIALS
DNA sample
Pipette tips and micropippetes
1x TAE or TBE buffer
Gel casting trays, combs and moulds
Electrophoresis power supply
Masking tape
6x loading dye
100bp ladder or lambda Hindlll marker
Conical flask
Agarose
Parafilm
RedSafe stain or Ethidium Bromide
UV transluminator
METHODS
Preparation of agarose gel
The percentage of agarose was determined to prepare.
The amount of agarose was calculated to be used and weight it on a balance using a weighing boat.
The agarose powder was dispenced into a conical flask.
An appropriate amount of TAE or TBA was measured using cylinder and poured it into the conical flak containing the agarose powder.
A piece of aluminium foil was used to cap the conical flask.
The mixture was heated in a microwave, hotplate or oven a Bunsen burner until it boils.
Once the agarose has dissolved, it was removed from the microwave and let it cool to about 50-60`c
Staining an agarose gel with RedSafe
The gel trays and comb was set up. A masking tape was used to seal the ends of the gel mould.
5 micro leter of RedSafe was added to 100ml agarose
It was mixed avoiding the formation of bubbles.
The agarose gel was poured into the gel mould.
It was allowed to solidify.
Staining an agarose gel with Ethidium Bromide.
Methods 1
The gel trays and comb was setuped.
5micro leter of ethidium bromide staining solusion was added to 100ml agarose.
The gel was allowed to solidify.
The agarose gel was run by placing the gel in the electrophoresis apparatus, filled the apparatus with 1x TAE or TBA until it covers the gel by about half a centimeter.
Method 2
This is the electrophoresis and to do so, the enough 0.5 µg/ml ethidium bromide in buffer or sterle water was prepared to completely submerge the gel. This solusion was stable for 1-2 months at room temperatire in the dark.
After running the gel, the gel was submerged in the staining solution for 15-30mins.
The gel was rocked back and forth gently occasionally.
The gel was placed on plastic/saran wrap on a UV transluminator.
Loading sample into wells of the agarose gel
A piese of parafilm was cut.
1µL of 6x was pipetted loading dye and it was placed on the parafilm.
5µL of plasmid was pipetted into the well of the agarose gel.
The mixture was gently loaded into the well of the agarose gel. The first well was kept empty for DNA marker.
The step 2-5 was repeated until all samples have been loaded into the gel.
3-7µL of DNA marker was loaded.
The gel was run at 100v for 40 mins or until the loading dye has migrated 75% of the gel.
The DNA was visualized on a UV transluminator or stain the gel with ethidium bromide.
The gel was photographed as instructed by our instructor
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