【摘要】：實驗一 pEGFP-C1質(zhì)粒的轉(zhuǎn)化、抽提及鑒定 目的:制備可供基因轉(zhuǎn)染所需的增強型綠色熒光蛋白質(zhì)粒pEGFP-C1。 方法:采用氯化鈣法制備E.coli JM109感受態(tài),將pEGFP-C1轉(zhuǎn)化此感受態(tài)菌,經(jīng)選擇性培養(yǎng)基篩選將陽性轉(zhuǎn)化菌落進行擴增。采用堿裂解法小量抽提質(zhì)粒,經(jīng)EcoR Ⅰ酶切鑒定為陽性克隆后再擴大培養(yǎng),用堿裂解法大量抽提,再次酶切鑒定并檢測質(zhì)粒的濃度和純度。 結(jié)果:感受態(tài)制備的實驗組細菌在含卡那霉素(Kanamycin)的瓊脂平板上形成了克隆,而對照組未能形成克隆。抽提的質(zhì)粒經(jīng)EcoR Ⅰ酶切后顯示一條帶,其大小為4.7kb,符合pEGFP-C1的長度。五次質(zhì)粒大量抽提的pEGFP-C1的A260/A280均在1.8～2.0間,濃度為0.87～1.75μg/μl間。 結(jié)論:1.氯化鈣法能有效地制備感受態(tài)菌,轉(zhuǎn)化pEGFP-C1質(zhì)粒的E.coli JM109表達Kanamycin抗性基因。2.抽提的質(zhì)粒經(jīng)酶切分析證實為pEGFP-C1。3.堿裂解法方法可靠,質(zhì)量穩(wěn)定,重復性好,大量抽提的pEGFP-C1濃度和純度均能滿足基因轉(zhuǎn)染的要求。 實驗二 羥基磷灰石納米粒的制備、表面修飾及與DNA的結(jié)合 目的:制備經(jīng)多聚賴氨酸表面修飾的羥基磷灰石納米粒,研究其與質(zhì)粒DNA結(jié)合的能力。
[Abstract]:Transformation, extraction and Identification of pEGFP-C1 plasmid objective: preparation of enhanced green fluorescent protein pEGFP-C1. for gene transfection Methods: the E.coli JM109 receptive state was prepared by calcium chloride method, and pEGFP-C1 was transformed into this strain. The positive transformed colony was amplified by selective medium screening. The plasmids were extracted by alkaline lysis method, identified as positive clones by EcoR 鈪，

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