Establishment and Optimization of ISSR-PCR Reaction System in Precocious Pyrus pyrifolia
Author(s): SONG Yan-chun, LI Yong-yu, CHEN Jian-yan, HUANG Tian-yi, GONG Li, WU Shao-hua
Pages: 91-96
Year: 2013 Issue: 2
Journal: Subtropical Plant Science
Keyword: precocity; Pyrus pyrifolia; ISSR; system optimization;
Abstract: To obtain 7 factors (DNA template dosage, Taq DNA polymerase dosage, primer concentration, dNTP concentration, Mg2+concentration, annealing temperature and PCR cycles ) of ISSR-PCR Reaction System in Pyrus pyrifolia ‘Cuiguan’, a precocious cultivar of southern China was been used as template. An optimum reaction system was been established. The final total volume was 20 μL, contains 2 μL 10×PCR buffer (Mg2+ free), 60 ng DNA template, 0.5 U Taq DNA polymerase, 1 μmol/L of primer concentration, 90 μmol/L of dNTP concentration, 2.25 mmol/L of Mg2+ concentration. Amplication procedure was as follows: initial-denaturing of 5 min at 94 ℃, followed by 42 cycles of 45 s for denaturing at 94℃, at annealing temperature for 45 s and 1 min at 72 ℃, after that 10 min extending at 72 ℃, and then keep at 4 ℃. Use 1.5% of agarose gel electrophoresis to test the genetic polymorphism.
Pages: 91-96
Year: 2013 Issue: 2
Journal: Subtropical Plant Science
Keyword: precocity; Pyrus pyrifolia; ISSR; system optimization;
Abstract: To obtain 7 factors (DNA template dosage, Taq DNA polymerase dosage, primer concentration, dNTP concentration, Mg2+concentration, annealing temperature and PCR cycles ) of ISSR-PCR Reaction System in Pyrus pyrifolia ‘Cuiguan’, a precocious cultivar of southern China was been used as template. An optimum reaction system was been established. The final total volume was 20 μL, contains 2 μL 10×PCR buffer (Mg2+ free), 60 ng DNA template, 0.5 U Taq DNA polymerase, 1 μmol/L of primer concentration, 90 μmol/L of dNTP concentration, 2.25 mmol/L of Mg2+ concentration. Amplication procedure was as follows: initial-denaturing of 5 min at 94 ℃, followed by 42 cycles of 45 s for denaturing at 94℃, at annealing temperature for 45 s and 1 min at 72 ℃, after that 10 min extending at 72 ℃, and then keep at 4 ℃. Use 1.5% of agarose gel electrophoresis to test the genetic polymorphism.
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