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Možnosti izolace nukleových kyselin v rostlinných a živočišných matricích
Truong, Thanh Huong
Isolation of nucleic acids is first step in most of molecule diagnosis. Literal abstract deals with various opportunities of nucleic acid isolation is the main part of this bachelor thesis. This work is about the most used methods from the oldest ones to the ones that are used nowadays. It covers methods such as salting in and salting out, chromatography, phenol -- chloroform extraction and isolation with magnetic particles. In the one of three chapters of literal abstract the basis of nucleic acids is described. Inhibitors for separation of nuclein acids are presented in another chapter of literal abstract. Minor part of the bachelor thesis deals with experimental process. The quality of the DNA acquired from various Cannabis sativa matrices are compared at two different weights of 20 mg and 100 mg. Evaluation of the results shows that use of 20mg oves us better quality.
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Povrchové modifikace paramagnetických částic pro izolaci nukleových kyselin
Vopařilová, Petra
The aim of the diploma thesis Surface modification of paramagnetic particles for nucleic acid isolation was to optimize the protocol for nucleic acid (NA) isolation using variously modified particles (MPs) so as to maximize the yield and quality of isolated NA and reduce the cost and time of this method. One of the most important requirements in selecting a suitable method of isolation is that the resulting NA can be used for subsequent applications. In my diploma thesis, the real-time PCR reaction was also optimized, in which RNA from isolations was tested. The theoretical part of the thesis describes the structure of MPs, their synthesis and their biotechnological use. Next, the basic methods for NA isolation are described. The isolation of NA using MPs and the possibility of automation of the method are described in more detail. In practical part the individual steps of isolation optimization are described. The results of these optimizations are also presented. In the practical part were tested MPs without surface modification, MPs whose surface was modified with tetraethoxysilane (TEOS), MPs whose surface was modified with tetraethoxysilane and then functionalized with (3-aminopropyl) -triethoxysilane (TEOS + APTES) and MPs on which surface have also been applied -COOH group (TEOS APTES COOH). The most suitable particles for RNA isolation appear to be modified MPs (TEOS + ATES), which enabled us to release almost all RNA in the primary elution, while a large amount of gDNA was released in the secondary elution. Thus, the isolation protocol could be universal for both gDNA isolation and RNA isolation, where in the first elution RNA would be released using an acidic elution solution (eg pH 6–6.6), in the secondary elution gDNA would be released using an alkaline solution (eg pH 7.5–8). MPs (TEOS) interact on their surface with the NA molecule probably only through a salt bridge, which forms chaotropic salts between the negatively charged phosphate groups and the negatively charged surface of the particles. MPs (TEOS + APTES) have better binding abilities, the interaction of NA with the surface of MPs is mediated not only through the salt bridge, but probably also by electrostatic interactions of positively charged amino groups (-NH3 +) of the APTES molecule with the negatively charged NA molecule.The resulting RNA isolated using MPs (TEOS + APTES) was compared to RNA isolated using a gravity column. The quality and integrity of the isolated RNA by MPs was sufficient for the real-time PCR reaction in which RNA was tested.
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