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Study of reversible adsorption of nucleid acids on magnetic carriers
Šálek, Petr ; Ing.Daniel Horák, CSc. (referee) ; Rittich, Bohuslav (advisor)
Reversible adsorption of nucleid acids on magnetic carriers was studied in this diploma thesis. Magnetic P(HEMA-co-GMA) microspheres and magnetic glass particles were used. The aim of the study was to isolate DNA in suitable quality for polymerase chain reaction (PCR). Adsorption of DNA on magnetic carriers was achieved after DNA condensation by PEG and NaCl in separation mixture. PEGs of various molecular weight (600 and 6000 g/mol) and different concentrations of PEG in separation mixture (4, 8, 12, 16%) were used. Quantity of eluted DNA incerased with molecular weight and concentration of PEG in separation mixtures. Optimized experimental conditions were applied for the separation of DNA from chicken erythrocytes, purified DNA, DNA in crude lysates of bacterial cells of Lactobacillus paracasei ssp. paracasei CCDM 211/06 and from real samples (liquid dairy products, hard cheese). The presence of target DNA in eluates was tested using genus specific PCR (genus Lactobacillus) or species specific PCR (species Bifidobacterium longum) Aqueous two-phase system (liquid-liquid) was used for separation of DNA from real symplex, too. At first the condiotions aqueous two-phase systém creation were studied. It was created by 16% PEG of various molecular weight (600, 6000 g/mol) and by various concentration of ammonium sulphate. Reversible DNA adsorption on carboxyl group-containing magnetic nonporous P(HEMA-co-EDMA) microspheres for the isolation PCR-ready DNA from liquid dairy products containing PCR inhibitors was studied, too. The quality of isolated DNA was checked by PCR amplification.The presumption on the elimination of PCR inhibitors from DNA samples was confirmed.
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Study of reversible adsorption of nucleid acids on magnetic carriers
Šálek, Petr ; Ing.Daniel Horák, CSc. (referee) ; Rittich, Bohuslav (advisor)
Reversible adsorption of nucleid acids on magnetic carriers was studied in this diploma thesis. Magnetic P(HEMA-co-GMA) microspheres and magnetic glass particles were used. The aim of the study was to isolate DNA in suitable quality for polymerase chain reaction (PCR). Adsorption of DNA on magnetic carriers was achieved after DNA condensation by PEG and NaCl in separation mixture. PEGs of various molecular weight (600 and 6000 g/mol) and different concentrations of PEG in separation mixture (4, 8, 12, 16%) were used. Quantity of eluted DNA incerased with molecular weight and concentration of PEG in separation mixtures. Optimized experimental conditions were applied for the separation of DNA from chicken erythrocytes, purified DNA, DNA in crude lysates of bacterial cells of Lactobacillus paracasei ssp. paracasei CCDM 211/06 and from real samples (liquid dairy products, hard cheese). The presence of target DNA in eluates was tested using genus specific PCR (genus Lactobacillus) or species specific PCR (species Bifidobacterium longum) Aqueous two-phase system (liquid-liquid) was used for separation of DNA from real symplex, too. At first the condiotions aqueous two-phase systém creation were studied. It was created by 16% PEG of various molecular weight (600, 6000 g/mol) and by various concentration of ammonium sulphate. Reversible DNA adsorption on carboxyl group-containing magnetic nonporous P(HEMA-co-EDMA) microspheres for the isolation PCR-ready DNA from liquid dairy products containing PCR inhibitors was studied, too. The quality of isolated DNA was checked by PCR amplification.The presumption on the elimination of PCR inhibitors from DNA samples was confirmed.
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Vliv rekombinantní bakterie Lactobacillus plantarum na vývoj pylové alergieL
Schwarzer, Martin ; Repa, A. ; Wiedermann, U. ; Hrnčíř, Tomáš ; Daniel, C. ; Pot, B. ; Štěpánková, Renata ; Hudcovic, Tomáš ; Tlaskalová, Helena ; Součková, Martina ; Kozáková, Hana
Germfree mice were monocolonized with recombinant Lactobacillus plantarum producing Bet v 1. Subsequently mice were sensitized by intraperitoneal injections to Bet v 1. We found out that monocolonization with recombinant L. plantarum reduced the level of specific Bet v 1 specific IgE antibody and stimulated production of INF-gamma in spleen cells supernatants. We conclude that monocolonization by recombinant L. plantarum shifts the immune response towards Th-1 direction and that it is a promising vaccine candidate against type I allergy
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