摘要
目的：筛选优化小鼠ＲＡＰＤ－ＰＣＲ反应体系及扩增程序。方法：以４０条引物对９个品系的小鼠基因组ＤＮＡ在各种不同条件下进行ＲＡＰＤ－ＰＣＲ，分析比较扩增结果。结果：在下列反应体系及扩增程序下结果较好：在２５μｌ的反应体系中含有１０×ｂｕｆｅｒ２．５μｌ，ｄＮＴＰ２００μｍｏｌ／Ｌ，Ｍｇ２＋２．５ｍｍｏｌ／Ｌ，引物０．２μｍｏｌ／Ｌ，Ｔａｑ酶１．５Ｕ，模板ＤＮＡ２０ｎｇ，扩增程序为９４℃变性１ｍｉｎ，３８℃退火１ｍｉｎ，７２℃延伸２ｍｉｎ，４０个循环后接１０ｍｉｎ延伸。结论：以上述反应体系及扩增程序进行小鼠ＲＡＰＤ－ＰＣＲ，扩增结果稳定。
Objective:
To optimize the RAPD PCR procedure in mice. Methods: Forty primers were used to amplify
DNA in 9 strains of mice and then the results of the amplification were analyzed. Results:
Optimal reaction system was as follows: 25 μl of each PCR mixture containing 2.5 μl of 10×
Taq Reaction buffer, 200 μmol/L of each dNTPs, 2.5 mmo/L of Mg 2+ , 0.2 μmol/L of primer,
1.5 unit of Taq DNA polymerase and 20 ng of total DNA. The reaction condition was 40 cycles of
denaturation at 94℃ for 1 min, annealing at 38℃ for 1 min and extension at 72℃ for 2 min.
Conclusion: The use of the above mentioned reaction system and amplifying procedure for
RAPD PCR in mice can get stable results.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
1999年第1期19-21,共3页
Journal of Third Military Medical University
基金
国家自然科学基金
全军"九五"医药卫生科研基金
重庆市攻关项目
