矽奈米微粒的組成及其於純化質體純化的應用

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標題:	矽奈米微粒的組成及其於純化質體純化的應用 Evaluation of Silica Nanocomposites for Efficient Extraction of Plasmid DNA from a Bacterial Cell Lysate
作者:	蕭閩誼
貢獻者:	嘉南藥理科技大學：生物科技系暨研究所王貞雅
關鍵字:	純化質體去氧核醣核酸四甲基矽氧烷胍鹽酸氨丙基三乙氧基矽烷四乙基矽氧烷 Plasmid DNA Silica particals Tetramethoxysilane Purification Aminopropyltriethoxysilane Tetraethoxysilane Guanidine hydrochloride
日期:	2009
上傳時間:	2011-10-27 14:43:14 (UTC+8)
摘要:	摘要早期純化質體 DNA 方法中，大多會使用到一些有機溶劑，例如，酚(Phenol)、氯仿 (Chloroform)，以上物質有可能殘留於樣品中，而影響往後之實驗。本實驗的目的利用鹼裂解緩衝溶液達到破菌之效果，並且利用溶膠-凝膠法 (Sol-Gel) 反應機制製備 TMOS、TEOS、TMOS/APTES、TEOS/APTES 之矽粒子，來純化質體 DNA。實驗的條件探討，會先選擇利用商業套組來純化質體 DNA，並將純化所得之質體 DNA，與所製備之矽粒子進行結合。然而，如何有效提供環境給予矽粒子吸附質體 DNA，就是實驗所探討之地方。提供之環境探討會選擇 GuHCl、CaCl2、NaCl、((NH4)2SO4) 以上之鹽類，探討鹽類濃度與 pH 值，結果發現實驗中以GuHCl、CaCl2 所提供環境最好。最後，會嘗試改變鹼裂解緩衝溶液之組成，改變之部分以中和溶液 (Solution III) 做條件探討，以 Ammonium acetate、Sodium acetate、ammonium sulfate、Tri-sodium citrate dihydrate、Potassium dihydrogen phosphate dehydrate、Potassium acetate 以上之鹽類做條件探討，其中發現以3M Potassium acetate/ 3.2M GuHCl pH5.8 矽粒子吸附質體 DNA效果最好。在將矽粒子所純化之質體 DNA，利用限制內切酶水解反應證明所純化之質體 DNA 適宜應用於後續之分生實驗用途。 Abstract The isolation of high quality, biologically active plasmid DNA is extremely important in the field of biotechnology. The early developed traditional of plasmid isolation method rely on phenol/chloroform technique. The method is classical but with some disadvantages such as toxicity and complexes operation. So there were several methods have been developed gradually to improve the isolation of plasmid DNA such as column chromatography, selective adsorption using solid phase supports and so on. Among these methods, solid phase supports to isolate plasmid DNA was commonly used for its simplicity and practicality. Silica has been widely selected as one of effective solid phases to purify plasmid DNA for its several features of straightforward synthesis, easily modification and biocompatibility. Silica matrix-based kits for rapid isolation of plasmid DNA are also commercially available (e.g., Invitrogen, Qiagen). The binding agents are indispensable by using of silica as solid phase for adsorption and isolation of plasmid DNA in the reported works. Though numerous methods that prepare hybrid silica particles for adsorbing DNA have been developed, the search for the best purification procedure and comparison of different materials in terms of the extraction efficiency of DNA has gained less attention. Our research was focused on testing different silica nanocomposites and binding agents in search of the best efficiency and plasmid-binding capacities for pDNA extraction. In this work, the pET28b plasmid has been successfully concentrated and purified from a broth culture of E. coli DH5α. The purified plasmid was then tested for purity and bioactivity through its use in a number of downstream applications.
關聯:	校內一年後公開，校外永不公開，學年度：97， 65 頁
显示于类别:	[生物科技系(所)] 博碩士論文

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