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DNA isolation using newly designed magnetic carriers
Machan, Radoslav ; Španová, Alena (referee) ; Rittich, Bohuslav (advisor)
Theoretical part of the master thesis was aimed on giving an overview of basic characteristics of magnetic particles, their morphology, basic methods of their synthesis, interaction with DNA and recent applications in biotechnology and biomedicine. The experimental part of thesis was aimed on application of new designed magnetic particles for isolation of both lactobacilli DNA and calf thymus DNA. Two types of magnetic beads were used: hyperbranched poly(glycidyl methacrylate-co-[2-(methacryloyloxy) ethoxy]acetic acid-co-ethylene dimethylacrylate) microbeads covered with amino groups (P(GMA-MOEAA-EDMA)-NH2) and magnetic non-porous poly(2-hydroxyethyl methacrylate) microbeads covered with carboxyl groups (P(HEMA-co-GMA)-COOH). For both types of microbeads two different protocols for preparation of separation mixtures with two different concentrations od poly(ethyleneglycol) 6000 (PEG 6000) as condensation agent were tested. Differences among both types of magnetic microbeads and DNAs used were found. It was shown that both types of microbeads are suitable for DNA isolation in the presence of 8% PEG 6000.
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DNA isolation using newly designed magnetic carriers
Machan, Radoslav ; Španová, Alena (referee) ; Rittich, Bohuslav (advisor)
Theoretical part of the master thesis was aimed on giving an overview of basic characteristics of magnetic particles, their morphology, basic methods of their synthesis, interaction with DNA and recent applications in biotechnology and biomedicine. The experimental part of thesis was aimed on application of new designed magnetic particles for isolation of both lactobacilli DNA and calf thymus DNA. Two types of magnetic beads were used: hyperbranched poly(glycidyl methacrylate-co-[2-(methacryloyloxy) ethoxy]acetic acid-co-ethylene dimethylacrylate) microbeads covered with amino groups (P(GMA-MOEAA-EDMA)-NH2) and magnetic non-porous poly(2-hydroxyethyl methacrylate) microbeads covered with carboxyl groups (P(HEMA-co-GMA)-COOH). For both types of microbeads two different protocols for preparation of separation mixtures with two different concentrations od poly(ethyleneglycol) 6000 (PEG 6000) as condensation agent were tested. Differences among both types of magnetic microbeads and DNAs used were found. It was shown that both types of microbeads are suitable for DNA isolation in the presence of 8% PEG 6000.
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Plasmid DNA vaccines
Machan, Radoslav ; Chroboková, Maria (referee) ; Rittich, Bohuslav (advisor)
Plasmid DNA vaccines are the new generation of vaccines with a great potential in prevention of many diseases. Recent studies and clinical test are aimed at prevention against cancer, hepatitis, malaria, HIV, influenza and other diseases. Recent main challenges covering plasmid DNA vaccines are associated with optimalization of each step of production and mainly purification steps allowing production of pDNA at kilogram levels. Main purification techniques used are based upon chromatographic methods, but research and development also shows other potential methods, like two-phase aqueous systems or magnetic microparticles as carriers. In experimental part of this thesis isolation of pUC 19 plasmid from Escherichia coli JM 109 (pUC 19) cell culture was performed via method of alkaline lysis. Isolation was verified by agarose gel electrophoresis. Isolated samples were purified in four repetitions with lithium chloride and magnetic microparticle carriers and the extent of purification was verified spectrophotometrically. Purified samples were visualised via agarose gel electrophoresis and results were compared.
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