4聚合酶链式反应.ppt

2004级应用生物教育专业 2005-4-12 2003级生物科学专业 2005-2006(上)分子生物学实验 二、实验原理(Experimental Principle) The polymerase chain reaction (PCR) is used to amplify a sequence of DNA using a pair of oligonucleotide primers each complementary to one end of the DNA target sequence. These are extended towards each other by athermostable DNA polymerase in a reaction cycle of three steps: denaturation, primer annealing and polymerization. The PCR cycle: (1) The reaction cycle comprises a 95°C step to denature the duplex DNA (2) an annealing step of around 55°C to allow the primers to bind (3) a 72°C polymerization step. Mg2+ and dNTPs are required in addition to template, primers, buffer and enzyme. A typical amplification reaction includes Primers (引物) Template DNA (模板DNA ) Taq DNA Polymerase (Taq DNA聚合酶) Nucleotides （寡核苷酸, dNTP） PCR buffer (PCR缓冲液) 三、试剂与器材（Reagents and apparatus） Ⅱ. Reagents 1. Sterile water 无菌水 2. 10×PCR buffer 10×PCR缓冲液 3. 25 mM MgCl2 氯化镁 4. 10 mM dNTPs 单核苷酸 5. 50μM oligonucleotide primers 1 677F 引物1 6. 50μM oligonucleotide primers 2 677R 引物2 7. 5 unit/μl Taq DNA polymerase Taq DNA 聚合酶 8. Template DNA 模板DNA 四、实验步骤(Experimental Procedures) 1.Combine the following for each reaction in a PCR tube (0.2ml） [在PCR (0.2ml）小管中将下列各组分混合]： 2. Prepare a control reaction with no template DNA and an addition 35.5μl of sterile water. [设立对照反应，其中不含模板DNA，而加35.5 μl 无菌水。] 3. Run the following program: [按以下程序运行] 95 ℃ preheated 8 min 95℃ 1 min. 62℃ 1 min. 72℃ 1 min for 36 cycles. Program a final extension at 72 ℃ for 7 min. PCR Amplifier Start Files Options Lid