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Optimization of calcium chloride concentration for removal of polysaccharide contamination during plant DNA isolation
Frnčová, Ekaterina ; Šlosárová, Katarína (referee) ; Fialová, Lenka (advisor)
The greatest difficulty in isolating DNA is the presence of contaminants that cause side effects. Polysaccharides are the most common contaminants in fruits. They can distort the results in spectrophotometric determination of purity or act as inhibitors in PCR analysis together with other substances (for example, proteins or phenolic substances). The aim of this work was to investigate the influence of different concentrations of calcium chloride on the process of DNA isolation. In the experimental part, DNA from the apple was isolated using different concentrations of calcium chloride. The isolation was carried out four times, and each time the sample was adjusted in different ways. It was found that the isolation method used works only with a sample that has been lyophilized. Isolation of DNA from fresh fruit provided very low yields. Probably, this was due to the large water content in the sample, and the proportion of the solid component was smaller. Subsequently, PCR analysis and electrophoresis were performed to determine the amplifiability of the isolated DNA. Two sets of primers with different specificity were used for this analysis. Amplifiability was confirmed only when using primers specific to apple DNA when using 100 mM solution of CaCl2. Other samples have been amplifiable using both types of primers. Probably, samples isolated using a 100 mM solution of CaCl2 had a larger amount of inhibitors that do not affect all PCR reactions equally, which may also indicate a small effectiveness of this amount of CaCl2.
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