The Building Of The System Of Competitor Quantitative PCR Of Heat Shock Protein 70 In Tribolium Castaneum

Using the RT-PCR skills we build the system of competitor quantitativePCR of the heat shock protein70 gene of Tribolium castaneum. The way to get thecompetitive contraposition template is: according to the gene sequence published onGeneBank, using computer softwares to select primers. In the appropriate temperature, wecan get the simple ribbon of the heat shock protein70 gene of Tribolium castaneum. Thenlower the temperature, we can get several ribbons and select one except for the one of theheat shock70 gene of Tribolium castaneum, finally, we get the pure plasmid of the oneselected. And the plasmid is the competitive contraposotion template of the system ofcompetitor quantitative PCR of the heat shock protein70 gene of Tribolium castaneum. Theplasmid and the the heat shock protein70 gene of Tribolium castaneum we get all have theprimer sequence, but the molecular weight of their PCR offspring are different ,so we candivide them easily .In the competitive PCR, we put a series diluent plasmid and the samequantum Heat shock70 gene of Tribolium castaneum to the same tube. Because they needthe same primer and the primer is limited, their quantum of PCR have interdependency.Using a kind of machine we can get the ratio of their PCR offspring, so we can draw thestandard curve and get the equation betweem the original concentration and the ratio oftheir PCR offspring, and the equation is Y=1.032X-1.618,and r2=0.975。 From the research we get the inside competitor and build the system of competitorquantitative PCR of the heat shock protein70 gene of Tribolium castaneum. So it is veryconvenient to study the quantum of the expression of heat shock70 gene of Triboliumcastaneum, and it is the foundation to study the expression regularity of heat shock70 gene. 51At the same time, it gives light to the theory of building the system of competitorquantitative PCR on insects.