自制实时定量PCR试剂在基因扩增中的效果评价

Effect Evaluation of Self-made Real-time Quantitative PCR Reagent in Gene Amplification

实验目的:评价自制实时定量PCR试剂在基因扩增效率上与商品化的差异,同时优化试剂配方,保证其稳定性。实验方法:1.原核表达纯化Taq DNA聚合酶,PCR确定纯化Taq DNA聚合酶的活性及在实时定量PCR中的使用浓度。2.自制实时定量PCR试剂,加入适当浓度的EvaGreen,以pAAV-LacZ质粒为模板,CMV为引物,进行实时定量聚合酶链反应,根据Ct值的变化,琼脂糖凝胶电泳,扩增曲线,溶解曲线来评估反应的灵敏度及特异性。3.调整自制的Real-time PCR mix中甘油,BSA等的浓度,在保证实时定量PCR结果准确性的情况下,尽可能的保证Taq DNA聚合酶的活性。

Objective:To evaluate the difference of gene amplification efficiency between self-made real-time quantitative PCR reagent and commercialization,and optimize the reagent formμla to ensure its stability.Experimental methods:1.Prokaryotic expression and purification of Taq DNA polymerase,PCR to determine the activity of purified Taq DNA polymerase and its concentration in real-time quantitative PCR.2.Self-made real-time quantitative PCR reagent,EvaGreen with proper concentration,pAAV-LacZ plasmid as template,CMV as primer,real-time quantitative polymerase chain reaction,according to the change of Ct value,agarose gel electrophoresis,amplification curve and dissolution curve to evaluate the sensitivity and specificity of the reaction.3.Adjust the concentration of glycerol and BSA in the self-made Real-time PCR mix to ensure the activity of Taq DNA polymerase as much as possible while ensuring the accuracy of real-time quantitative PCR resμlts.