摘要
为建立鹅掌楸简单序列重复区间扩增ISSR的PCR优化反应体系,以鹅掌楸叶片基因组DNA为材料,系统地测试了模板DNA、引物、dNTPs、Mg2+浓度、TaqDNA聚合酶用量及退火温度对ISSR-PCR反应体系的影响。结果表明:优化的PCR反应体系为:20μL总体系中,含30 ng模板DNA,0.3μmol.L-1随机引物,0.2 mmol.L-1dNTPs,1.4 mmol.L-1Mg2+,0.8 UTaqDNA聚合酶;最佳退火温度为60℃;PCR反应程序为:94℃预变性5 min;94℃变性1 min,60℃退火45 s,72℃延伸2 min;45个循环;72℃再延伸7 min。
In order to establish the optimal system of ISSR-PCR for Liriodendron,with the genomic DNA of Liriodendron leaves as material,the parameters including template DNA,primers,dNTPs,Mg2+ concentration,dose of Taq DNA polymerase and annealing temperature to ISSR-PCR were systematically analyzed.The results show that the optimal reaction system of ISSR(20 μL) was: 30 ng template DNA,0.6 μmol·L-1 random primers,0.2 mmol·L-1 dNTPs,1.4 mmol·L-1 Mg2+,and 0.8 U Taq DNA polymerase;and that the optimized annealing temperature was 60℃;the PCR procedure was: pre-denaturizing at 94℃ for 5 min,denaturizing at 94℃ for 1 min,annealing at 60℃ for 45 s,extension at 72℃ for 2 min,reaction of 45 cycles and re-extension at 72℃ for 7 min.
出处
《中南林业科技大学学报》
CAS
CSCD
北大核心
2010年第6期80-84,共5页
Journal of Central South University of Forestry & Technology
基金
湖南省林业厅重点项目(2007-10)