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乳液PCR法扩增复杂基因序列方法的建立.rar

资料分类:农业大学 高级会员(姐妹花)提供原创论文资料更新时间:2016-01-21
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摘要:目的 基于PCR技术的基本原理分析了普通PCR方法对于扩增复杂基因序列的不足,通过实验研究,建立乳液PCR方法以期解决这一问题。方法 采用以人工合成的乳酸杆菌质粒为模板,不同成分配比条件下的乳液PCR和普通PCR方法对质粒序列进行特异性的有效扩增,并用PCR扩增产物进行琼脂糖凝胶电泳,电泳结束后对凝胶进行Gel red染色,使用GS-800灰度扫描仪成像。分析了乳液PCR和普通PCR扩增产物的特异性以及不同成分配比条件下乳液PCR的扩增质量。结果 通过两种不同PCR方法的结果对比,发现在相同条件不同温度下,乳液PCR扩增特异性大于普通PCR扩增反应,并确定了在25 oC时,当水相中加入100g/L BSA,水油体积比在1:2.5时,破乳水饱和乙醚体积比为1:1时,水油相混合在5min,1700rpm条件下得到了最佳的扩增效果。

关键词:乳液PCR;普通PCR;BSA;破乳

 

Abstract:Objective Based on general principle of PCR analysis the shortage of Amplification Complex Gene Sequence by Conventional PCR, here we construct a protocol to minimize these problems. Methods Plasmid as a template with Lactobacillus, different components of the emulsion PCR and conventional PCR plasmid template amplification effectively, and increases the product with PCR to carry on agarose electrophoresis. After the electrophoresis had finished, carries on the Gel red dyeing to the gelatin, uses GS-800 gradation scanner image formation.Then analysis emulsion PCR and conventional PCR amplification products' specificity.To obtain In situation of that different components of the emulsion PCR products quality. Results Through contrasting with two methods , Emulsion PCR amplification specificity is stronger than conventional PCR in the same conditions. Summarize on the condition of 25 oC, add 100g/l BSA to aqueous phase, volume ratio of water-in-oil(w/o) at 1:2.5, volume ratio of breaking water-saturated diethyl ether at 1:1. Water and oil mixture by thoroughly mixing the following components in 5min,1700rpm would got the best amplification effect.

Key words:Emulsion PCR; Conventional PCR; BSA; Break emulsion

 

论文资料贡献者对本文的描述:分析了乳液PCR和普通PCR扩增产物的特异性以及不同成分配比条件下乳液PCR的扩增质量。结果 通过两种不同PCR方法的结果对比,发现在相同条件不同温度下,乳液PCR扩增特异性大于普通......
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