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Comparison of gel and capillary electrophoresis of proteins
Krbec, Michal ; Franěk, Tomáš (advisor) ; Kotaška, Karel (referee)
Electrophoresis of serum proteins is one of the methods used in laboratory diagnosis of pathological conditions, but also for research purposes. This is a one-step method, which involves isolation of individual proteins from a mixture (mostly blood proteins) in the electric field in the presence of a suitable buffer and a suitable carrier based on their different mobility, dependent on their surface charge and molecular weight. Originally the carrieer used to be a paper, later cellulose acetate membrane and now it is almost exclusively agarose (purified agar with low endoosmosis) or polyacrylamide (PAA). My task in this work was to analyze a serie of samples by gel electrophoresis and subsequently analyze the same serie by capillary electrophoresis. Results obtained by both methods I worked numerically, compared with each other and statistically evaluated. The output of my work is a comparison of gel and capillary electrophoresis, evaluation of the advantages, disadvantages and especially the differences between these two methods.
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The interactions of hyaluronan and DNA
Sklenářová, Renáta ; Krouská, Jitka (referee) ; Hurčíková, Andrea (advisor)
This diploma thesis deals with the study of possible interactions between hyaluronan (HA) and plasmid DNA (pDNA). Plasmid DNA was isolated from E. coli JM109 (pUC19) and resuspended in TE buffer as well as high molecular weight hyaluronan. Individual samples of pDNA, HA and pDNA-HA were characterized by gel electrophoresis, CD spectroscopy and high resolution ultrasonic spectroscopy. Agarose gel electrophoresis examined the effect of the addition of hyaluronan to plasmid DNA on the migration of samples to the positive electrode. Structural changes in pDNA-HA samples were examined using CD spectroscopy. Individual CD spectra describes the dependence of the difference in absorption coefficients for left-hand and right-handed elliptic polarized light at wavelength. High resolution ultrasonic spectroscopy has been used to study interactions. It is an analytical method based on ultrasonic velocity and attenuation. We classify this technique as a non-destructive method because the passing waves do not affect the structure of the analyzed sample.
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Preprocessing of 1D gel electrophoresis image
Hlavatý, Matej ; Harabiš, Vratislav (referee) ; Vítek, Martin (advisor)
The aim of this project is an automatic analysis of 1D gel electrophoresis results. Basic principles of gel electrophoresis and types of errors and signal noise influencing the result are studied. The proposed algorithm serves for automatic detection of lanes in the image. Summation projection on the horizontal axis is created from black-and-white digital pictures of electrophoresis. Local minima of the profile are located after smoothing the profile. In the location of these minima borders between lanes are established.
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Automatic improvements of images from 1D gel electrophoresis
Kovář, Martin ; Vítek, Martin (referee) ; Škutková, Helena (advisor)
In this bachelor’s thesis are explained the basic principles of electrophoresis and its modalities with focusing on 1D gel electrophoresis. It describes analysis of an electrophoreogram and causes of its possible distortion, and states specifications of applications of the method in microbiology, genomics and proteomics. The practical part presents development, optimization and outputs of a programme for automatic electrophoreogram analysis, which was created in Matlab environment. The analysis contains lane and band detection and computation of samples’ molecular weight. The ending of the thesis is constituted by evaluating efficiency of detection and accuracy of weight computation.
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Plasmid DNA interactions with lanthanoide compounds
Gőghová, Sabína ; Španová, Alena (referee) ; Rittich, Bohuslav (advisor)
In the theoretical part of bachelor thesis, literature was summarised on the isolation and purification of plasmid DNA, the application of lanthanides as non enzymatic nucleases and practical importance of lanthanide compounds in medicine. In the experimental part of the thesis, plasmid DNA pUC19 was isolated by the method of alkaline lysis. Isolated plasmid DNA was cleaved by neodymium trichloride at 70 °C in different intervals. Gel electrophoresis was used for evaluation of experiments.
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Plasmid DNAs interactions with lanthanoide compounds
Budko, Kateryna ; Horák, Daniel (referee) ; Rittich, Bohuslav (advisor)
Recently much attention is given to lanthanides and their complexes as excellent catalysts for cleavage of nucleic acids. The thesis has been focused on the cleavage of plasmid and bacterial DNA by ions Nd3+ and Y3+ and by different carriers containing the lanthanide compounds. The creation of single-stranded nicks and double-stranded ones in the plasmid DNA molecules was studied by agarose gel electrophoresis. Verification of the cleavage of bacterial DNA was made by polymerase chain reaction using primers specific for the domain Bacteria and genus and species-specific primers. The results will be used in the development of the method that will allow perfect carriers`s coverage verification with the magnetic perovskit nucleus and other carriers with the lanthanide compounds.
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Classification of 1D gel electrophoresis samples
Krupka, Ondřej ; Škutková, Helena (referee) ; Vítek, Martin (advisor)
This term project deals with the classification of 1D gel electrophoresis samples. It describes the theoretical information about gel electrophoresis, various types of errors, processing of the image and its classification using the cluster analysis. One of the main goals is creation of images with the highest quality as possible. A realization of pre-processing and detection of the sample borders is made in the MATLAB environment. And finally, classification of samples is done with subsequent statistical analysis.
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Electrophoretic and immunofluorescence methods for study of plant cell cultures
Klimešová, Marie ; Babula, Petr (referee) ; Provazník, Ivo (advisor)
In all organisms are rising a reactive oxygen and nitrogen species by the effects of various stress factors and these species have a negative impact on the organism. Due to this species plants have built up an efficient antioxidant system, that helps them to resist negative effects of reactive oxygen and nitrogen species. In this work was researched the effect of hydrogen peroxide and sodium benzoate on the production of hydrogen peroxide, superoxide, reactive nitrogen species and malondialdehyde, contained in the root and above-ground part of maize (Zea mays L.). By use of the fluorescence microscopy there were obtained images of cross-cut of root from which was determined the intensity of fluorescence of individual parts of the root and was examined the effect of the intensity of fluorescence markers of oxidative stress in dependence on the type of the fluorescence filter used.
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Preparation and purification of plasmid DNAs
Němeček, Milan ; Španová, Alena (referee) ; Rittich, Bohuslav (advisor)
With the development of therapeutic methods in medicine, like are DNA vaccines and gene therapy increases demand for new processes for the isolation and purification highly pure plasmid DNA. Most often used methods of purification plasmid DNA are chromatographic methods. In experimental part of this thesis was performed isolation of plasmid pUC-19 DNA plasmid via alkalyne lysis. And purification of plasmid was performed by liquid chromatography and agarose gel electrophoresis.
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Polymorphism of microsatellite markers in selected \kur{Beauveria bassiana} strains/isolates
KRÁLOVÁ, Martina
\kur{Beauveria bassiana} is a entomopathogenic polyphagous fungus commonly found in soil and it is parasite of soil insects, mainly of the stages of insect that occur in soil. At the present time it is used in plant protection against more than 70 species of insects. In the Czech Republic \kur{Beauveria bassiana} has the greatest importance in the fight against bark beetle \kur{Ips typographus} in the NP Šumava in these days. This study was focused on the evaluation of genetic variability \kur{Beauveria bassiana} strains on the basis of microsatellite analysis and the comparison of four separation methods: electrophoresis in 2% agarose gel, electrophoresis in 3% synergel, chip electrophoresis and fluorescent capillary electrophoresis in term of the most precise separation of PCR products. We used 41 strains which were collected in the NP Šumava and 20 strains from long-term collection determined as an exotic in this study. This large geographical scale group contains the strains from whole world and in addition it was upgraded by the strains collected from the NP Krkonoše and South Moravia. For the microsatellite analysis there were used 11 pairs of primers but for inter-comparison of separative methods were chosen only 4 pairs of primers. The population of \kur{Beauveria bassiana} strains collected from the NP Šumava were evaluated by analysis of microsatellites as a conservative and fully closed regardless of the source and the location. The strains from the large geographical scale group showed the great genetic variability. In terms of separation, the best and most suitable separation method was proved, the fluorescent capillary electrophoresis. Despite of its difficult financial aspect, this method was evaluated as the most precise and the most sensitive. Its advantage is in possibility to detect the smallest differences in the length of single allele in the range 1-2 bp, which is for the gel electrophoresis impossible.
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