Optimization of ISSR-PCR Reaction System and Primer Screening of Halerpestes cymbalaris
The genome DNA of Halerpestes cymbalaris was extracted by the improved CTAB method.The orthogonal design method was used to the ISSR-PCR system of H.cymbalarisat four levels of five factors (the concentration of Taq DNA polymerase,dNTPs,primer,template DNA and magnesium ion).The results were analyzed using the range analysis and variance analysis.A suitable system of the ISSR-PCR reaction of H.cymbalaris was established which contained 0.025 U/μL Taq polymerase,1.5 mmol/L magnesiumion,1.0 mmol/L dNTPs,0.5 μmol/L primer and 1.5 ng/μL template DNA in total 20 μL reaction.Ten ISSR primers with clear bands were selected.And the annealing temperature of each primer was determined by the gradient PCR.On these bases,the optimum cycles were determined.The optimum cycles were 40 times.
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万方数据
维普
