In this chapter agarose gel electrophoresis will be introduced that is one of the foremost frequently used separation technic in laboratories working with nucleic acids.
The basics of the technology dealing with foreign protein expression in bacteria are also discussed in this chapter.
For these, background information can be found here on:
Agarose gel electrophoresis
Gene expression regulation in bacteria
Bacterial expression systems Hands on practice will be:
Preparation and running of agarose gel
Analysis of plasmid quality by gel electrophoresis
Transformation of expression plasmid to specific host cells
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Part I: Agarose gel electrophoresis
Theoretical background
Agarose gel electrophoresis is suitable for the separation and size determination of DNA fragments. This method is suitable for the analysis, identification and isolation of DNA fragments. By standard gel electrophoresis, 0.2-50 kb sized DNA fragments can be easily separated although different agarose concentrations (0.8 to 3 %) are optimal for different ranges. By dissolving agarose in a specific buffer upon heating, linear molecules of polysaccharide polymer form a molecular mesh, which has comparable pore sizes to that of DNA fragments. When this gel is placed into electric field, charged molecules will move towards the oppositely charged pole. In practice agarose gels are placed in a horizontal arrangement by submerging the gel in a slightly alkaline buffer. DNA fragments (generally a mixture of those) are loaded into wells created close to the negative pole and when the electricity turned on the negatively charged DNA fragments migrate (run) towards the positively charged anode. In order to detect the position of fragments, fluorescent intercalating dye, most frequently ethidium bromide, is added to the gel. It is important to know that ethidium bromide (EtBr) is a mutagenic agent, since it can intercalate between the base pairs of the DNA! You should be careful with handling those solutions and gels, which contain this dye, therefore always use gloves and lab-coat, and pay attention to avoid contamination of work bench, pipettes and other laboratory equipment with EtBr. Due to the fluorescence of the EtBr under UV light, even a few ng of DNA could be detected. Gel images
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can be recorded by using UV-light camera assemble for the analysis of results obtained from gel electrophoresis. The sizes of DNA fragments can be determined based on their mobility on the gel by comparing that to the mobilities of fragments with known sizes, called DNA ladder.
DNA can also be isolated from the agarose gel and used for further experiments. In fact, this is the only way by which specific fragments from a mixture of fragments can be recovered for the usage of further genetic engineering.
Preparation of an agarose gel
First, the appropriate amount of agarose should be dissolved in electrophoresis buffer. For this, the required amount of agarose should be placed in a flask and after that the appropriate buffer should be added to it (the order is important!). Then the suspension should be heated until the agarose is completely dissolved (until the solution is clean and transparent). Since the inhalation of evaporated EtBr is very dangerous, the solution should be cooled down until it is hand-warm before the addition of it. After that the appropriate amount of EtBr should be added to the solution, then it should be mixed well but carefully to avoid bubble formation. As a next step, the EtBr containing solution should be poured into a gel tray with a comb placed in the correct position. When the agarose is cooled down, the gel is whitish-coloured. Gel can be kept for several hours wrapped in folia or overlapped with buffer or it can be used immediately.
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Factors influencing the migration of DNA fragments in agarose gel:
1. Electric field
DNA fragments are moved within the gel according to the electric potential. In slightly alkaline or neutral buffer the negatively charged DNA fragments migrate towards the positive pole. Electrophoresis can be performed at a wide-range of voltage gradient (0.25-7 V/cm). The higher voltage naturally results in quicker migration, but it also decreases the resolution of the gel and very high current could melt and destroy the gel.
In practice 5 V/cm is the most frequently used voltage.
2. DNA size:
In gels, larger DNA molecules migrate slower then shorter ones.
The explanation of this is that for larger molecules it is more difficult to get through the pores of the gel. Note, that the driving force of the movement, such as the voltage difference between the poles of the power supply, exerts the same dragging force to a unique size of DNA fragment independently of the size of it, since the density of charges uniformly distributes in the polymer.
3. DNA structure:
Relaxed circular, linear and superhelical circular DNA molecules consisting of the same nucleotides in identical order (such as topological isomers of the same DNA) migrate in the agarose matrix with different rates (see in Chapter 3).
92 4. Electrophoretic buffer:
The mobility of the DNA also depends on the compounds and the ion concentration of the electrophoretic buffer. If the ion concentration is too high (e.g. if somebody uses 50x buffer instead of 1x), the current will be too high, thereby leading to high amount of heat generation. In the worst case the gel could melt, therefore the DNA could be denatured.
5. Agarose concentration:
The density of the agarose gel determines the sizes of the pores.
Gels with different concentrations have different separation ranges:
Amount of agarose Separation range (to linear dsDNA)
0.7 % 0.8-12 kb
1.0 % 0.5-10 kb
1.5 % 0.2-3 kb
2.0 % 0.1-2 kb
Table 4.1 Relationship between agarose concentration and the separation range of agarose gel
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