摘要
以甘肃金鳟尾鳍为试验材料,提取基因组DNA。对影响甘肃金鳟RAPD扩增的重要参数进行了优化试验,包括模板DNA浓度、dNTP浓度、引物浓度、Taq酶、变性时间、退火温度等,建立了甘肃金鳟RAPD反应的最适体系。即在25μl反应体系中:模板DNA用量为4.0 ng/μl;Mg2+浓度为2.5mmol/L;dNTP浓度为0.5 mmol/L;引物浓度为0.5μmol/L;Taq酶用量为1 U;扩增程序:94℃预变性4 min,94℃变性45 s,36℃退火50 s,72℃延伸1 min,共35个循环,最后延伸10 min。
The DNA was extracted from the caudal fin of Gansu rainbow trout Oncrohychus mykiss, and the conditions for RAPD analysis were optimized, including DNA concentration, dNTPs concentration, primer concentration, denaturation time and annealing temperature. Then the RAPD reaction system was established. The reactions were performed in a volume of 25 μl containing 2.5 μl 10 × Buffer, 2.5 mmol/L Mg^2＋ , Taq DNA ploymerase 1U, 0.5 mmol/L dNTPs, 0.5 μmol/L Primer, and 4.0 ng/μl template DNA. PCR program was 4 min at 94 ℃ for predenaturation, then 35 cycles of 45 s at 94 ℃ for denaturation,50 s at 36℃ for annealing, 1 min at 72 ℃ for extension, finally extension at 72℃ for 10 min.
出处
《水产科学》
CAS
北大核心
2008年第7期352-355,共4页
Fisheries Science
基金
甘肃省自然科学基金资助项目(0710RJZA096)
关键词
甘肃金鳟
RAPD反应
体系构建
优化
Gansu Oncrohychus mykiss
RAPD
reaction system
optimization
