Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/3502
標題: 離子效應對質體核酸純化影響之研究
作者: 姜禮明
關鍵字: 質體核酸
離子效應
出版社: 化學工程學系
摘要: 本研究是將溶離後的細胞先用1.0M胍基鹽酸與50%異丙醇作純化前處理,再使用陰離子交換樹脂 Q Sepharose,進行批次及流動式純化操作實驗。在批次操作中,用不同離子(包括陽離子Na+、K+、Mg2+、Ca2+與陰離子SO42-、PO43-、CH3COO-)作為清洗溶液,固定異丙醇濃度,改變溶液中之離子強度去分離不純物。探討離子效應對質體核酸純化時的影響。 在批次操作時,發現陽離子純化後的產率及純度受到異丙醇濃度的影響很大。當30﹪iPrOH時,除了鈣離子之外,隨著清洗液離子濃度的增加,產率會降低,但純度會提高。45﹪iPrOH時,不管鹽類濃度如何增加,都不太容易將質體核酸洗出。所以1.0M NaCl/45﹪iPrOH才有接近100﹪產率及100﹪純度。三種陰離子(硫酸根、磷酸根及醋酸根離子),在批次操作時,有不錯的產率,約80﹪左右,但純度都不是100﹪。 流動式操作時選擇氯化鎂及醋酸鈉作為清洗液藉由降低清洗液中異丙醇的濃度,來達到純化的效果。在小量操作時,固定清洗液中MgCl2濃度為1.5M,發現異丙醇為20﹪時會清洗出純度100%的質體核酸。醋酸鈉的情形亦同,固定清洗液中CH3COONa濃度為2.0M,在20﹪的異丙醇濃度時會清洗出純度100%的質體核酸,但產率只有47%左右。中量操作和小量操作的結果類似。在大量純化時我們只選擇用氯化鎂作為清洗液,純化7.8kb的質體核酸pCMV-βgal發現產率降低,約只有20﹪左右,但純度還是100%。純化不同大小的質體核酸,產率會有差別但純度還是維持100%。 我們證實前處理後再進行質體核酸的純化可得高純度的質體核酸,樹脂經過再生程序後可重複使用。並不影響質體核酸的純度。
In this study, the cell after alkaline lysis, 1.0M guanidine-Cl and 50% isopropanol was added in the pretreatment. Then plasmid DNA purification was used anion exchange chromatography in batch and gravity-flow experiments. In batch experiment, we used washing buffer contain different kind of metal ion include cation: Na+、K+、Mg2+、Ca2+and anion: SO42-、PO43-、CH3COO- to eliminate contaminate. The effects of ions were examined for purification of plasmid DNA. In batch experiment the concentration of isopropanol would affect the yield and purity in purification. When washing buffer contain 30% isopropanol, added more salt, the yield of elution plasmid DNA reduced, but purity would raise, expect Ca2+ washing buffer. If the isopropanol was 45% in washing buffer, no matter how much salt added, the plasmid DNA could not wash out. The best condition of washing buffer was 1.0M NaCl/45% isopropanol/50mM MOPS, the purity of plasmid DNA was 100% and the yield was approach 100%. When washing buffer contain anion ion example :SO42-、PO43-、CH3COO-, we could obtain about 80% yield, but the purity was almost not good. We use MgCl2 and CH3COONa as washing buffer with step-down isopropanol concentrations in gravity flow method. In small scale, after purification of 7.5 ml of fermentation broth , the yield of MgCl2 and CH3COONa washing buffer were 63﹪±1 and 47﹪±2. In minimum scale(300ml broth) the yield of MgCl2 and CH3COONa washing buffer were 71﹪±8 and 20﹪±2. When purification process was scale up to 3000 ml broth, we use MgCl2 washing buffer only. The yield of 4.7kb and 7.8 Kb plasmid was 61﹪±1and 20﹪,respectively. The purity of all scales was good. We demonstrated lysised cell after pretreatment could obtain high purity of plasmid DNA. After generation the matrix could reuse. The purity after purification was not affected.
URI: http://hdl.handle.net/11455/3502
Appears in Collections:化學工程學系所

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