摘要
在ＲＡＰＤ反应中，有许多因素影响结果的稳定性和准确性．该文选取了菊属６种植物，对ＲＡＰＤ各种反应条件进行了摸索．结果表明：菊属植物较为理想的反应体系为：２０μｌ反应体积含１０ｍｍｏｌ／ｌＴｒｉｓ－ＨＣｌ（ｐＨ为８．３），５０ｍｍｏｌ／ｌＫＣｌ，２ｍｍｏｌ／ｌＭｇＣｌ２，０．００１％ｇｅｌａｔｉｎ，２００μｍｏｌ／ｌｄＮＴＰ，５０～２００ｎｇ引物，０．７５～１．０单位（Ｕ）ＴａｑＤＮＡｐｏｌｙｍｅｒａｓｅ，２～１０ｎｇ基因组ＤＮＡ．在２０个核苷酸引物情况下，反应条件为：９２℃、３ｍｉｎ预变性；９２℃、４５ｓ，５０℃、１ｍｉｎ，７２℃、２ｍｉｎ，循环４０周；最后一次延伸为７２℃、１０ｍｉｎ．应用上述反应体系进行扩增反应，反应产物在２％琼脂糖凝胶上以５Ｖ／ｃｍ电场强度电泳２～４ｈ。
Many factors of the RAPD reaction procedure may influence the results. This paper presents selected RAPD reaction condition for analysis of genetic relationship among Dendranthema species. A more extensive study of influential factors is also presented. To get good results, the reaction should be performed in a volume of 20μl containing 10mmol/lTris-HCl(pH8. 3), KCI 50mmol/l, MgCl2 2mmol/l, gelatin 0. 001%, dNTP 200μmol/l,primer 50￣200ng, Taq DNA polymerase 0. 75￣1. 0 unit, genomic DNA 2￣10ng. The mixture was laid on thermal cycle. After 3 minutes at 92C, 40 cycles were run. Each cycle consisted of 45 seconds at 92℃, 1 minute at 50℃, 2 minutes at 72℃. Then 10 minutes at 72℃.Plant growth state, methods of DNA isolation were also discussed as influential factors.
出处
《北京林业大学学报》
CAS
CSCD
北大核心
1996年第1期46-51,共6页
Journal of Beijing Forestry University
基金
国家自然科学基金
关键词
菊属
RAPD
反应体系
Dendranthema, RAPD reaction condition
